Antibodies specific for native PrPSc

ABSTRACT

Antibodies are disclosed which specifically bind to native PrP Sc  in situ. Preferred antibodies bind only to the native PrP Sc  of a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrP Sc . Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrP Sc , (3) neutralizes the infectivity of prions, (4) bind to PrP Sc  in situ and (5) bind 50% or more of PrP Sc  in a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrP c  denatured via proteinase K) for the presence of PrP Sc  of a specific species which PrP Sc  is associated with disease. Antibodies which specifically bind to human PrP Sc  can be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease. The antibodies are preferably produced using phage display technology wherein the genetic material in the phage expressing the antibody is obtained from a mammal with an ablated endogenous PrP protein gene and an endogenous chimeric PrP gene which mammal had been inoculated with PrP Sc  to induce antibody production.

This application is a divisional application of Ser. No. 08/713,939,filed Sep. 13, 1996, now U.S. Pat. No. 5,846,533 which is acontinuation-in-part application of Ser. No. 08/528,104, filed Sep. 14,1995 (now abandoned), both of which are incorporated herein by referencein their entirety and to which applications we claim priority tinder 35USC § 120.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Grant No. AGO 2132 awarded by the National Institutes ofHealth.

FIELD OF THE INVENTION

This invention relates to methods for obtaining antibodies and assaysfor using such antibodies. More specifically, the invention relates tomethods of obtaining antibodies which specifically bind to naturallyoccurring forms of PrP^(Sc).

BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause central nervous systemspongiform encephalopathies in humans and animals. Prions are distinctfrom bacteria, viruses and viroids. The predominant hypothesis atpresent is that no nucleic acid component is necessary for infectivityof prion protein. Further, a prion which infects one species of animal(e.g., a human) will not infect another (e.g., a mouse).

A major step in the study of prions and the diseases that they cause wasthe discovery and purification of a protein designated prion protein(“PrP”) [Bolton et al., Science 218:1309-11 (1982); Prusiner, et al.,Biochemistry 21:6942-50 (1982); McKinley, et al., Cell 35:57-62 (1983)].Complete prion protein-encoding genes have since been cloned, sequencedand expressed in transgenic animals. PrP^(C) is encoded by a single-copyhost gene [Basler, et al., Cell 46:417-28 (1986)] and is normally foundat the outer surface of neurons. Prion diseases are accompanied by theconversion of PrP^(C) into a modified form called PrP^(Sc). However, theactual biological or physiological function of PrP^(C) is not known.

The scrapie isoform of the prion protein (PrP^(Sc)) is necessary forboth the transmission and pathogenesis of the transmissibleneurodegenerative diseases of animals and humans. See Prusiner, S. B.,“Molecular biology of prion disease,” Science 252:1515-1522 (1991). Themost common prion diseases of animals are scrapie of sheep and goats andbovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. andWells, Microbiol. Immunol. 172:21-38 (1991)]. Four prion diseases ofhumans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease(CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatalfamilial insomnia (FFI) [Gajdusek, D. C., Science 197:943-960 (1977);Medori et al., N. Engl. J. Med. 326:444-449 (1992)]. The presentation ofhuman prion diseases as sporadic, genetic and infectious illnessesinitially posed a conundrum which has been explained by the cellulargenetic origin of PrP.

Most CJD cases are sporadic, but about 10-15% are inherited as autosomaldominant disorders that are caused by mutations in the human PrP gene[Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al., Science258:806-808 (1992); Kitamoto et al., Proc. R. Soc. Lond. (In press)(1994)]. Iatrogenic CJD has been caused by human growth hormone derivedfrom cadaveric pituitaries as well as dura mater grafts [Brown et al.,Lancet 340:24-27 (1992)]. Despite numerous attempts to link CJD to aninfectious source such as the consumption of scrapie infected sheepmeat, none has been identified to date [Harries-Jones et al., J. Neurol.Neurosurg. Psychiatry 51:1113-1119 (1988)] except in cases ofiatrogenically induced disease. On the other hand, kuru, which for manydecades devastated the Fore and neighboring tribes of the New Guineahighlands, is believed to have been spread by infection duringritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases ofthe Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (NewYork: Academic Press), pp. 66-90 (1979)].

The initial transmission of CJD to experimental primates has a richhistory beginning with William Hadlow's recognition of the similaritybetween kuru and scrapie. In 1959, Hadlow suggested that extractsprepared from patients dying of kuru be inoculated into non-humanprimates and that the animals be observed for disease that was predictedto occur after a prolonged incubation period [Hadlow, W. J., Lancet2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpersdemonstrated the transmissibility of kuru to chimpanzees afterincubation periods ranging form 18 to 21 months [Gajdusek et al., Nature209:794-796 (1966)]. The similarity of the neuropathology of kuru withthat of CJD [Klatzo et al., Lab Invest. 8:799-847 (1959)] promptedsimilar experiments with chimpanzees and transmissions of disease werereported in 1968 [Gibbs, Jr. et al., Science 161:388-389 (1968)]. Overthe last 25 years, about 300 cases of CJD, kuru and GSS have beentransmitted to a variety of apes and monkeys.

The expense, scarcity and often perceived inhumanity of such experimentshave restricted this work and thus limited the accumulation ofknowledge. While the most reliable transmission data has been said toemanate from studies using non-human primates, some cases of human priondisease have been transmitted to rodents but apparently with lessregularity [Gibbs, Jr. et al., Slow Transmissible Diseases of theNervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York:Academic Press), pp. 87-110 (1979); Tateishi, et al., Prion Diseases ofHumans and Animals, Prusiner, et al., eds. (London: Ellis Horwood), pp.129-134 (1992)].

The infrequent transmission of human prion disease to rodents has beencited as an example of the “species barrier” first described by Pattisonin his studies of passaging the scrapie agent between sheep and rodents[Pattison, I. H., NINDB Monograph 2, D. C. Gajdusek, C. J. Gibbs Jr. andM. P. Alpers, eds. (Washington, D. C.: U.S. Government Printing), pp.249-257 (1965)]. In those investigations, the initial passage of prionsfrom one species to another was associated with a prolonged incubationtime with only a few animals developing illness. Subsequent passage inthe same species was characterized by all the animals becoming ill aftergreatly shortened incubation times.

The molecular basis for the species barrier between Syrian hamster (SHa)and mouse was shown to reside in the sequence of the PrP gene usingtransgenic (Tg) mice [Scott, et al., Cell 59:847-857 (1989)]. SHaPrPdiffers from MoPrP at 16 positions out of 254 amino acid residues[Basler, et al., Cell 46:417-428 (1986); Locht, et al., Proc. Natl.Acad. Sci. USA 83:6372-6376 (1986)]. Tg(SHaPrP) mice expressing SHaPrPhad abbreviated incubation times when inoculated with SHa prions. Whensimilar studies were performed with mice expressing the human, or ovinePrP transgenes, the species barrier was not abrogated, i.e., thepercentage of animals which became infected were unacceptably low andthe incubation times were unacceptably long. Thus, it has not beenpossible, for example in the case of human prions, to use transgenicanimals (such as mice containing a PrP gene of another species) toreliably test a sample to determine if that sample is infected withprions. The seriousness of the health risk resulting from the lack ofsuch a test is exemplified below.

More than 45 young adults previously treated with HGH derived from humanpituitaries have developed CJD [Koch, et al., N. Enql. J. Med.313:731-733 (1985); Brown, et al., Lancet 340:24-27 (1992); Fradkin, etal., JAMA 265:880-884 (1991); Buchanan, et al., Br. Med. J. 302:824-828(1991)]. Fortunately, recombinant HGH is now used, although theseemingly remote possibility has been raised that increased expressionof wtPrP^(C) stimulated by high HGH might induce prion disease[Lasmezas, et al., Biochem. Biophys. Res. Commun. 196:1163-1169 (1993)].That the HGH prepared from pituitaries was contaminated with prions issupported by the transmission of prion disease to a monkey 66 monthsafter inoculation with a suspect lot of HGH [Gibbs, Jr., et al., N.Enql. J. Med. 328:358-359 (1993)]. The long incubation times associatedwith prion diseases will not reveal the full extent of iatrogenic CJDfor decades in thousands of people treated with HGH worldwide.Iatrogenic CJD also appears to have developed in four infertile womentreated with contaminated human pituitary-derived gonadotrophin hormone[Healy, et al., Br. J. Med. 307:517-518 (1993); Cochius, et al., Aust.N.Z. J. Med. 20:592-593 (1990); Cochius, et al., J. Neurol. Neurosurc.Psychiatry 55:1094-1095 (1992)] as well as at least 11 patientsreceiving dura mater grafts [Nisbet, et al., J. Am. Med. Assoc. 261:1118(1989); Thadani, et al., J. Neurosurg. 69:766-769 (1988); Willison, etal., J. Neurosurg. Psychiatric 54:940 (1991); Brown, et al., Lancet340:24-27 (1992)]. These cases of iatrogenic CJD underscore the need forscreening pharmaceuticals that might possibly be contaminated withprions.

Recently, two doctors in France were charged with involuntarymanslaughter of a child who had been treated with growth hormonesextracted from corpses. The child developed Creutzfeldt-Jakob Disease.(See New Scientist, Jul. 31, 1993, page 4). According to the PasteurInstitute, since 1989 there have been 24 reported cases of CJD in youngpeople who were treated with human growth hormone between 1983 andmid-1985. Fifteen of these children have died. It now appears as thoughhundreds of children in France have been treated with growth hormoneextracted from dead bodies at the risk of developing CJD (see NewScientist, Nov. 20, 1993, page 10.) Prior attempts to create PrPmonoclonal antibodies have been unsuccessful (see Barry and Prusiner, J.of Infectious Diseases Vol. 154, No. 3, Pages 518-521 (1986). Thus thereis a need for an assay to detect compounds which result in disease.Specifically, there is a need for a convenient, cost-effective assay fortesting sample materials for the presence of prions which cause CJD. Thepresent invention offers such an assay.

SUMMARY OF THE INVENTION

Antibodies of the invention will specifically bind to a native prionprotein (i.e., native PrP^(Sc)) in situ with a high degree of bindingaffinity. The antibodies can be placed on a substrate and used forassaying a sample to determine if the sample contains a pathogenic formof a prion protein. The antibodies are characterized by one or more ofthe following features (1) an ability to neutralize infectious prions,(2) will bind to prion proteins (PrP^(Sc)) in situ i.e., will bind tonaturally occurring forms of a prion protein in a cell culture or invivo and without the need to treat (e.g., denature) the prion protein,and (3) will bind to a high percentage of the PrP^(Sc) form (i.e.disease form) of prion protein in a composition e.g., will bind to 50%or more of the PrP^(Sc) form of the prion proteins. Preferred antibodiesare further characterized by an ability to (4) bind to a prion proteinof only a specific species of mammals e.g., bind to human prion proteinand not prion protein of other mammals.

An important object is to provide antibodies which bind to native prionprotein (PrP^(Sc)).

Another object is to provide antibodies which specifically bind toepitopes of prion proteins (PrP^(Sc)) of a specific species of animaland not to the prion protein (PrP^(Sc)) of other species of animals.

Another object is to provide monoclonal antibodies which specificallybind to prion proteins (PrP^(Sc)) associated with disease, (e.g., humanPrP^(Sc)) which antibodies do not bind to denatured PrP proteins notassociated with disease (e.g., human PrP^(C)).

Still another object is to provide specific methodology to allow othersto generate a wide range of specific antibodies characterized by theirability to bind one or more types of prion proteins from one or morespecies of animals.

Another object of the invention is to provide an assay for the detectionof PrP^(Sc) forms of PrP proteins.

Another object of the invention is to provide an assay which canspecifically differentiate prion protein (PrP^(Sc)) associated withdisease from PrP^(Sc) not associated with disease.

Another object is to detect prions which specifically bind to nativePrP^(Sc) of a specific species such as a human, cow, sheep, pig, dog,cat or chicken.

An advantage of the invention is that it provides a fast, efficient costeffective assay for detecting the presence of native PrP^(Sc) in asample.

A specific advantage is that the assay can be used as a screen for thepresence of prions (i.e., PrP^(Sc)) in products such as pharmaceuticals(derived from natural sources) food, cosmetics or any material whichmight contain such prions and thereby provide further assurances as tothe safety of such products.

Another advantage is that the antibodies which can be used with aprotease which denatures PrP^(c) thereby providing for a means ofdifferentiating between infectious (PrP^(Sc)) and non-infectious forms(PrP^(Sc)) of prions.

Yet another advantage of the invention is that antibodies of theinvention are characterized by their ability to neutralize theinfectivity of naturally occurring prions e.g., neutralize PrP^(Sc).

Another advantage is that antibodies of the invention will bind to(PrP^(Sc)) prion proteins in situ. i.e., will bind to naturallyoccurring (PrP^(Sc)) prions in their natural state in a cell culture orin vivo without requiring that the prion proteins be particularlytreated, isolated or denatured.

Another advantage is that the prion proteins of the invention will bindto a relatively high percentage of the infectious form of the prionprotein (e.g., PrP^(Sc))—for example bind to 50% or more of the PrP^(Sc)form of prion proteins in a composition.

An important feature of the invention is that the methodology makes itpossible to create a wide variety of different prion protein antibodieswith the same or individually engineered features which features maymake the antibody particularly suitable for uses such as (1) prionneutralization to purify a product, (2) the extraction of prion proteinsand (3) therapies.

A feature of the invention is that it uses phage display libraries inthe creation of the antibodies.

Another feature of the invention is that the phage are geneticallyengineered to express a specific binding protein of an antibody on theirsurface.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the chimeric gene, assay method, and transgenic mouse as morefully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a portion of PrP proteins showing thedifferences between a normal, wild-type human PrP protein and a normal,wild-type mouse PrP protein;

FIG. 2 shows the amino acid sequence of mouse PrP (SEQ ID NO:1) alongwith specific differences between mouse PrP (SEQ ID NO:1) and human PrP(SEQ ID NO:2);

FIG. 3 shows the amino acid sequence (SEQ ID NO:1) of mouse PrP andspecifically shows differences between mouse PrP (SEQ ID NO:1) andbovine PrP (SEQ ID NO:3);

FIG. 4 shows the amino acid sequence of mouse PrP and specifically showsdifferences between mouse PrP and ovine PrP (SEQ ID NO:4);

FIG. 5 is a bar graph of serum dilution vs optical density at 405 nm forthe mouse (D7282) for serum against denatured mouse PrP 27-30;

FIG. 6 shows the amino acid sequences of selected (A) heavy chain and(B) light chain variable regions generated by panning an IgG1 libraryfrom mouse D7282 against denatured MoPrP 27-30 rods;

FIG. 7 shows the deduced amino acid sequences for some of the phageclones obtained in one panning against PrP;

FIGS. 8A-8H show photos of histoblots 8A, 8B, 8C, 8D, 8E, 8F, 8G and 8Hshowing staining of SHaPrP 27-30 and denatured SHaPrP 27-30;

FIG. 9 is a graph showing the ELISA reactivity of purified Fabs againstprion protein SHa 27-30;

FIG. 10 is a graph of the ELISA reactivity of purified Fabs againstdenatured prion protein SHa 27-30;

FIG. 11 is a photo showing amino precipitation of SHaPrP 27-30 withrecombinant Fabs of the invention; and

FIG. 12 is a photo showing amino precipitation of SHaPrP 27-30 withpurified Fabs of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present antibodies, assays and methods for producing an usingsuch are disclosed and described, it is to be understood that thisinvention is not limited to particular antibodies, assays or method assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The terms “PrP protein”, “PrP” and the like are used interchangeablyherein and shall mean both the infectious particle form PrP^(Sc) knownto cause diseases (spongiform encephalopathies) in humans and animalsand the non-infectious form PrP^(C) which, under appropriate conditionsis converted to the infectious PrP^(Sc) form.

The terms “prion”, “prion protein” and “PrP^(Sc) protein” and the likeused interchangeably herein to refer to the infectious PrP^(Sc) form ofa PrP protein and is a contraction of the words “protein” and“infection” and the particles are comprised largely if not exclusivelyof PrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions include those which infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats as well as bovine spongiformencephalopathies (BSE) or mad cow disease and feline spongiformencephalopathies of cats. Four prion diseases known to affect humans are(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used—andin particular in humans and in domesticated farm animals.

The term “PrP gene” is used herein to describe genetic material whichexpresses proteins as shown in FIGS. 2-4 and polymorphisms and mutationssuch as those listed herein under the subheading “Pathogenic Mutationsand Polymorphisms.” The term “PrP gene” refers generally to any gene ofany species which encodes any form of a prion protein. Some commonlyknown PrP sequences are described in Gabriel et al., Proc. Natl. Acad.Sci. USA 89:9097-9101 (1992) which is incorporated herein by referenceto disclose and describe such sequences. The PrP gene can be from anyanimal including the “host” and “test” animals described herein and anyand all polymorphisms and mutations thereof, it being recognized thatthe terms include other such PrP genes that are yet to be discovered.The protein expressed by such a gene can assume either a PrP^(C)(non-disease) of PrP^(Sc) (disease) form.

The terms “standardized prion preparation”, “prion preparation”,“preparation” and the like are used interchangeably herein to describe acomposition containing prions (PrP^(Sc)) which composition is obtainedfrom brain tissue of mammals which contain substantially the samegenetic material as relates to prions, e.g., brain tissue from a set ofmammals which exhibit signs of prion disease which mammals (1) include atransgene as described herein; (2) have an ablated endogenous prionprotein gene; (3) have a high copy number of prion protein gene from agenetically diverse species; or (4) are hybrids with an ablatedendogenous prion protein gene and a prion protein gene from agenetically diverse species. The mammals from which standardized prionpreparations are obtained exhibit clinical signs of CNS dysfunction as aresult of inoculation with prions and/or due to developing the diseasedue to their genetically modified make up, e.g., high copy number ofprion protein genes.

The term “artificial PrP gene” is used herein to encompass the term“chimeric PrP gene” as well as other recombinantly constructed geneswhich when included in the genome of a host animal (e.g., a mouse) willrender the mammal susceptible to infection from prions which naturallyonly infect a genetically diverse test mammal, e.g., human, bovine orovine. In general, an artificial gene will include the codon sequence ofthe PrP gene of the mammal being genetically altered with one or more(but not all, and generally less than 40) codons of the natural sequencebeing replaced with a different codon—preferably a corresponding codonof a genetically diverse mammal (such as a human). The geneticallyaltered mammal being used to assay samples for prions which only infectthe genetically diverse mammal. Examples of artificial genes are mousePrP genes encoding the sequence as shown in FIGS. 2, 3 and 4 with one ormore different replacement codons selected from the codons shown inthese Figures for humans, cows and sheep replacing mouse codons at thesame relative position, with the proviso that not all the mouse codonsare replaced with differing human, cow or sheep codons. Artificial PrPgenes can include not only codons of genetically diverse animals but mayinclude codons and codon sequences not associated with any native PrPgene but which, when inserted into an animal render the animalsusceptible to infection with prions which would normally only infect agenetically diverse animal.

The terms “chimeric gene,” “chimeric PrP gene”, “chimeric prion proteingene” and the like are used interchangeably herein to mean anartificially constructed gene containing the codons of a host animalsuch as a mouse with one or more of the codons being replaced withcorresponding codons from a genetically diverse test animal such as ahuman, cow or sheep. In one specific example the chimeric gene iscomprised of the starting and terminating sequence (i.e., N- andC-terminal codons) of a PrP gene of a mammal of a host species (e.g. amouse) and also containing a nucleotide sequence of a correspondingportion of a PrP gene of a test mammal of a second species (e.g. ahuman). A chimeric gene will, when inserted into the genome of a mammalof the host species, render the mammal susceptible to infection withprions which normally infect only mammals of the second species. Thepreferred chimeric gene disclosed herein is MHu2M which contains thestarting and terminating sequence of a mouse PrP gene and a non-terminalsequence region which is replaced with a corresponding human sequencewhich differs from a mouse PrP gene in a manner such that the proteinexpressed thereby differs at nine residues.

The term “genetic material related to prions” is intended to cover anygenetic material which effects the ability of an animal to becomeinfected with prions. Thus, the term encompasses any “PrP gene”,“artificial PrP gene”, “chimeric PrP gene” or “ablated PrP gene” whichterms are defined herein as well as modification of such which effectthe ability of an animal to become infected with prions. Standardizedprion preparations are produced using animals which all havesubstantially the same genetic material related to prions so that all ofthe animals will become infected with the same type of prions and willexhibit signs of infection at about the same time.

The terms “host animal” and “host mammal” are used to describe animalswhich will have their genome genetically and artificially manipulated soas to include genetic material which is not naturally present within theanimal. For example, host animals include mice, hamsters and rats whichhave their PrP gene ablated i.e., rendered inoperative. The host isinoculated with prion proteins to generate antibodies. The cellsproducing the antibodies are a source of genetic material for making aphage library. Other host animals may have a natural (PrP) gene or onewhich is altered by the insertion of an artificial gene or by theinsertion of a native PrP gene of a genetically diverse test animal.

The terms “test animal” and “test mammal” are used to describe theanimal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, horse,cat, dog or chicken, and one may wish to determine whether a particularsample includes prions which would normally only infect the test animal.

The terms “genetically diverse animal” and “genetically diverse mammal”are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

The terms “ablated PrP protein gene”, “disrupted PrP gene”, and the likeare used interchangeably herein to mean an endogenous PrP gene which hasbeen altered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative. Examples of non-functional PrP genes andmethods of making such are disclosed in Büeler, H., et al “Normaldevelopment of mice lacking the neuronal cell-surface PrP protein”Nature 356, 577-582 (1992) and Weisman (WO 93/10227). The methodologyfor ablating a gene is taught in Capecchi, Cell 51:503-512 (1987) all ofwhich are incorporated herein by reference. Preferably both alleles ofthe genes are disrupted.

The terms “hybrid animal”, “transgenic hybrid animal” and the like areused interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having an ablated endogenous prionprotein gene with a second animal which includes either (1) a chimericgene or artificial PrP gene or (2) a PrP gene from a genetically diverseanimal. For example a hybrid mouse is obtained by cross-breeding a mousewith an ablated mouse gene with a mouse containing (1) human PrP genes(which may be present in high copy numbers) or (2) chimeric genes. Theterm hybrid includes any offspring of a hybrid including inbredoffspring of two hybrids provided the resulting offspring is susceptibleto infection with prions with normal infect only a genetically diversespecies. A hybrid animal can be inoculated with prions and serve as asource of cells for the creation of hybridomas to make monoclonalantibodies of the invention.

The terms “susceptible to infection” and “susceptible to infection byprions” and the like are used interchangeably herein to describe atransgenic or hybrid test animal which develops a disease if inoculatedwith prions which would normally only infect a genetically diverse testanimal. The terms are used to describe a transgenic or hybrid animalsuch as a transgenic mouse Tg(MHu2M) which, without the chimeric PrPgene, would not become infected with a human prion but with the chimericgene is susceptible to infection with human prions.

By “antibody” is meant an immunoglobulin protein which is capable ofbinding an antigen. Antibody as used herein is meant to include theentire antibody as well as any antibody fragments (e.g. F(ab′)₂, Fab′,Fab, Fv) capable of binding the epitope, antigen or antigenic fragmentof interest.

Antibodies of the invention are immunoreactive or immunospecific for andtherefore specifically and selectively bind to a PrP^(Sc) protein.Antibodies which are immunoreactive and immunospecific for natural ornative PrP^(Sc) are preferred. Antibodies for PrP^(Sc) are preferablyimmunospecific—i.e., not substantially cross-reactive with relatedmaterials. Although the term “antibody” encompasses all types ofantibodies (e.g., monoclonal) the antibodies of the invention arepreferably produced using the phage display methodology describedherein.

By “purified antibody” is meant one which is sufficiently free of otherproteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody “preferentially binds” to a native PrP^(Sc)protein (or an antigenic fragment thereof), i.e., does not substantiallyrecognize and bind to other antigenically-unrelated molecules. Apurified antibody of the invention is preferably immunoreactive with andimmunospecific for a PrP^(Sc) protein of specific species and morepreferably immunospecific for native human PrP^(Sc).

By “antigenic fragment” of a PrP protein is meant a portion of such aprotein which is capable of binding an antibody of the invention.

By “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide i.e., epitope of aPrP^(Sc) protein. Antibody binding to its epitope on this specificpolypeptide is preferably stronger than binding of the same antibody toany other epitope, particularly those which may be present in moleculesin association with, or in the same sample, as the specific polypeptideof interest e.g., binds more strongly to PrP^(Sc) than denaturedfragments of PrP^(C) so that by adjusting binding conditions theantibody binds almost exclusively to PrP^(Sc) and not denaturedfragments of PrP^(C). Antibodies which bind specifically to apolypeptide of interest may be capable of binding other polypeptides ata weak, yet detectable, level (e.g., 10% or less of the binding shown tothe polypeptide of interest). Such weak binding, or background binding,is readily discernible from the specific antibody binding to thecompound or polypeptide of interest, e.g. by use of appropriatecontrols. In general, antibodies of the invention which bind to nativePrP^(Sc) in situ with a binding affinity of 10⁷ mole/l or more,preferably 10⁸ mole/liters or more are said to bind specifically toPrP^(Sc). In general, an antibody with a binding affinity of 10⁶mole/liters or less is not useful in that it will not bind an antigen ata detectable level using conventional methodology currently used.

By “detectably labeled antibody”, “detectably labeled anti-PrP” or“detectably labeled anti-PrP fragment” is meant an antibody (or antibodyfragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels may be selected from a variety of such labels known inthe art, but normally are radioisotopes, fluorophores, paramagneticlabels, enzymes (e.g., horseradish peroxidase), or other moieties orcompounds which either emit a detectable signal (e.g., radioactivity,fluorescence, color) or emit a detectable signal after exposure of thelabel to its substrate. Various detectable label/substrate pairs (e.g.,horseradish peroxidase/diaminobenzidine, avidin/streptavidin,luciferase/luciferin)), methods for labelling antibodies, and methodsfor using labeled antibodies are well known in the art (see, forexample, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e., arresting its development; or

(c) relieving the disease, i.e., causing regression of the disease. Theinvention is directed toward treating patients with infectious prionsand is particularly directed toward treating humans infected withPrP^(Sc), resulting in a disease of the central nervous system such asbovine spongiform encephalopathy; Creutzfeldt-Jakob Disease; fatalfamilial insomnia or Gerstmann-Strassler-Scheinker Disease.

Abbreviations used herein include:

CNS for central nervous system;

BSE for bovine spongiform encephalopathy;

CJD for Creutzfeldt-Jakob Disease;

FFI for fatal familial insomnia;

GSS for Gerstmann-Strassler-Scheinker Disease;

Hu for human;

HuPrP for a human prion protein;

Mo for mouse;

MoPrP for a mouse prion protein;

SHa for a Syrian hamster;

SHaPrP for a Syrian hamster prion protein;

Tg for transgenic;

Tg(SHaPrP) for a transgenic mouse containing the PrP gene of a Syrianhamster;

Tg(HuPrP) for transgenic mice containing the complete human PrP gene;

Tg(ShePrP) for transgenic mice containing the complete sheep PrP gene;

Tg(BovPrP) for transgenic mice containing the complete cow PrP gene;

PrP^(Sc) for the scrapie isoform of the prion protein;

PrP^(C) for the cellular contained common, normal isoform of the prionprotein;

MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;

MHu2M for a chimeric mouse/human PrP gene wherein a region of the mousePrP gene is replaced by a corresponding human sequence which differsfrom mouse PrP at 9 codons;

Tg(MHu2M) mice are transgenic mice of the invention which include thechimeric MHu2M gene;

MHu2MPrP^(Sc) for the scrapie isoform of the chimeric human/mouse PrPgene;

PrP^(CJD) for the CJD isoform of a PrP gene;

Prnp^(0/0) for ablation of both alleles of an endogenous prion proteingene, e.g., the MoPrP gene;

Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) of transgenicmice expressing SHaPrP, +/0 indicates heterozygous;

Tg(HuPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a human prion protein gene (HuPrP) with a mouse with both allelesof the endogenous prion protein gene disrupted;

Tg(MHu2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a chimeric prion protein gene (MHu2M) with a mouse with bothalleles of the endogenous prion protein gene disrupted.

FVB for a standard inbred strain of mice often used in the production oftransgenic mice since eggs of FVB mice are relatively large and toleratemicroinjection of exogenous DNA relatively well.

GENERAL ASPECT OF THE INVENTION

The core of the invention is an antibody which specifically binds to aPrP^(Sc) protein and preferably binds to a native non-denatured PrP^(Sc)protein in situ with an affinity of 10⁷ moles/liter or more, preferable10⁸ moles/liter or more of a single species (e.g., human) and morepreferably binds only to human PrP^(Sc) and not denatured fragments ofhuman PrP^(C)). The antibody may bind to all proteins coded by thedifferent mutations and/or polymorphisms of the PrP protein gene.Alternatively, a battery of antibodies (2 or more different antibodies)are provided wherein each antibody of the battery specifically binds toprotein coded by a different mutation or polymorphism of the PrP gene.The antibody can be bound to support surface and used to assay a samplein vitro for the presence of a particular type of human PrP^(Sc). Theantibody can also be bound to a detectable label and injected into ananimal to assay in vivo for the presence of a particular type of nativePrP^(Sc).

Although there are known procedures for producing antibodies from anygiven antigen practice has shown that it is particularly difficult toproduce antibodies which bind to certain proteins e.g., PrP^(Sc). Thedifficulty with obtaining antibodies to PrP^(Sc) relates, in part, toits special and unknown qualities. By following procedures describedherein antibodies which bind native PrP^(Sc) in situ have been obtainedand others may follow the procedures described here to obtain otherantibodies to PrP^(Sc) and to other proteins for which it is difficultto generate antibodies.

To produce antibodies of the invention it is preferable to begin withinoculating a host mammal with prion proteins i.e., infectious PrP^(Sc).The host mammal may be any mammal and is preferably a host mammal of thetype defined herein such as a mouse, rat, guinea pig or hamster and ismost preferably a mouse. The host animal is inoculated with prionproteins which are endogenous to a different species which is preferablya genetically diverse species. For example a mouse is inoculated withhuman prion proteins. Preferably, the host mammal is inoculated withinfectious prion proteins of a genetically diverse mammal. For example,a mouse is inoculated with human PrP^(Sc). Using a normal host mammal inthis manner it is possible to elicit the generation of some antibodies.However, when a hosts animal includes a prion protein gene and isinoculated with prions from a genetically diverse species antibodieswill, if at all, only be generated for epitopes which differ betweenepitopes of the prion protein of the host animal and epitopes of thegenetically diverse species. This substantially limits the amount ofantibodies which might be generated and decreases the ability to find anantibody which selectively binds to an infectious form of a prionprotein and does not bind to denatured fragments of a non-infectiousform. Thus, unless one is attempting to generate antibodies whichdifferentiate between prion proteins of different species it ispreferable to begin the antibody production process using a mammal whichhas an ablated prion protein gene i.e., a null PrP gene abbreviated asPrnp^(0/0). Accordingly, the invention is generally described inconnection with the use of such “null” mammals and specificallydescribed in connection with “null mice.”

Antibodies are produced by first producing a host animal (e.g., a mouse)which has its endogenous PrP gene ablated, i.e., the PrP gene renderedinoperative. A mouse with an ablated PrP gene is referred to as a “nullmouse”. A null mouse can be created by inserting a segment of DNA into anormal mouse PrP gene and/or removing a portion of the gene to provide adisrupted PrP gene. The disrupted gene is injected into a mouse embryoand via homologous recombination replaces the endogenous PrP gene.

The null mouse is injected with prions in order to stimulate theformation of antibodies. Further, injections of adjuvants and prions aregenerally used to maximize the generation of antibodies.

The mouse is then sacrificed and bone marrow and spleen cells areremoved. The cells are lysed, RNA is extracted and reversed transcribedto cDNA. Antibody heavy and light chains (or parts thereof) and thenamplified by PCR. The amplified cDNA library may be used as is or aftermanipulation to create a range of variants and thereby increase the sizeof the library.

An IgG phage display library is then constructed by inserting theamplified cDNA encoding IgG heavy chain and the amplified cDNA encodinga light chain into a phage display vector (e.g., a pComb3 vector) suchthat one vector contains a cDNA insert encoding a heavy chain fragmentin a first expression cassette of the vector, and a cDNA insert encodinga light chain fragment in a second expression cassette of the vector.

Ligated vectors are then packaged by filamentous phage M13 using methodswell known in the art. The packaged library is then used to infect aculture of E. coli, so as to amplify the number of phage particles.After bacterial cell lysis, the phage particles are isolated and used ina panning procedure.

The library created is panned against a composition containing prions.Antibody fragments which selectively bind to PrP^(Sc) e.g., humanPrP^(Sc) are then isolated.

Specifics of a PrP Protein

The major component of purified infectious prions, designated PrP 27-30,is the proteinase K resistant core of a larger native protein PrP^(Sc)which is the disease causing form of the ubiquitous cellular proteinPrP^(C). PrP^(Sc) is found only in scrapie infected cells whereasPrP^(C) is present in both infected and uninfected cells implicatingPrP^(Sc) as the major, if not the sole, component of infectious prionparticles. Since both PrP^(C) and PrP^(Sc) are encoded by the samesingle copy gene, great effort has been directed toward unraveling themechanism by which PrP^(Sc) is derived from PrP^(C). Central to thisgoal has been the characterization of physical and chemical differencesbetween these two molecules. Properties distinguishing PrP^(Sc) fromPrP^(C) include low solubility (Meyer, et al 1986 PNAS), poorantigenicity (Kascack, J. Virol 1987; Serban D. 1990) proteaseresistance (Oesch, et al 1985 Cell) and polymerization of PrP 27-30 intorod-shaped aggregates which are very similar, on the ultrastructural andhistochemical levels, to the PrP amyloid plaques seen in scrapiediseased brains (Prusiner, et al Cell 1983). By using proteinase K it ispossible to denature PrP^(C) but not PrP^(Sc). To date, attempts toidentify any post-transitional chemical modifications in PrP^(C) thatlead to its conversion to Prs have proven fruitless (Stahl, et al 1993Biochemistry). Consequently, it has been proposed that PrP^(C) andPrP^(Sc) are in fact conformational isomers of the same molecule.

Conformational description of PrP using conventional techniques has beenhindered by problems of solubility and the difficulty in producingsufficient quantities of pure protein. However, PrP^(C) and PrP^(Sc) areconformationally distinct. Theoretical calculations based upon the aminoacid sequences of PrPs from several species have predicted four putativehelical motifs in the molecule. Experimental spectroscopic data wouldindicate that in PrP^(C) these regions adopt α-helical arrangements,with virtually no β-sheet (Pan, et al PNAS 1993). In dramatic contrast,in the same study it was found that PrP^(Sc) and PrP 27-30 possesssignificant β-sheet content, which is typical of amyloid proteins.Moreover, studies with extended synthetic peptides, corresponding to PrPamino acid residues 90-145, have demonstrated that these truncatedmolecules may be converted to either α-helical or β-sheet structures byaltering their solution conditions. The transition of PrP^(C) to PrPSCrequires the adoption of β-sheet structure by regions that werepreviously α-helical.

In general, scrapie infection fails to produce an immune response, withhost organisms being tolerant to PrP^(Sc) from the same species.Polyclonal anti-PrP antibodies have though been raised in rabbitsfollowing immunization with large amounts of SHaPrP 27-30 (Bendheim, etal PNAS 1985, Bode, et al J. Gen. Virol. 1985). Similarly, a handful ofanti-PrP monoclonal antibodies have been produced in mice (Kascack, etal, J. Virol. 1987, Barry, et al, J. Infect. Dis. 1986). Theseantibodies are able to recognize native PrP^(C) and denatured PrP^(Sc)from both SHa and humans equally well, but do not bind to MoPrP.Unsurprisingly, the epitopes of these antibodies were mapped to regionsof sequence containing amino acid differences between SHa- and MoPrP(Rogers, et al, J. Immunol. 1993).

It is not entirely clear as to why antibodies of the type described inthe above cited publications will bind to PrP^(C) but not to PrP^(Sc).Without being bound to any particular theory it is suggested that suchmay take place because epitopes which are exposed when the protein is inthe PrP^(C) conformation are unexposed or partially hidden in thePrP^(sc) configuration—where the protein is relatively insoluble andmore compactly folded together. It is pointed out that stating that anantibody binds to PrP^(C) but not to PrP^(Sc) is not correct in absoluteterms (but correct in commonly accepted terms) because some minimalbinding to PrP^(Sc) may occur. For purposes of the invention anindication that no binding occurs means that the equilibrium or affinityconstant K_(a) is 10⁶ l/mole or less. Further, binding will berecognized as existing when the K_(a) is at 10⁷ l/mole or greaterpreferably 10⁸ l/mole or greater. The binding affinity of 10⁷ l/mole ormore may be due to (1) a single monoclonal antibody (i.e., large numbersof one kind of antibodies) (2) a plurality of different monoclonalantibodies (e.g., large numbers of each of five different monoclonalantibodies) or (3) large numbers of polyclonal antibodies. It is alsopossible to use combinations or (1)-(3).

Preferred antibodies will bind 50% or more of the PrP^(Sc) in a sample.However, this may be accomplished by using several different antibodiesas per (1)-(3) above. It has been found that an increased number ofdifferent antibodies is more effective in binding a larger percentage ofthe PrP^(Sc) in a sample as compared to the use of a single antibody.For example, the use of six copies of a single antibody “Q” might bind40%. of the PrP^(Sc) in a sample. Similar results might be obtained withsix copies of antibody “R” and “S”. However, by using two copies each of“Q”, “R” and “S” the six antibodies will bind over 50% of the PrP^(Sc)in a sample. Thus, a synergistic effect can be obtained by combiningcombinations of two or more antibodies which bind PrP^(Sc) i.e., bycombining two or more antibodies which have a binding affinity K_(a) forPrP^(Sc) of 10⁷ l/mole or more. Thus combination of D4, R2, 6D2, D14, R1and R10 and/or related antibodies can provide synergistic results.

Antibody/Antigen Binding Forces

The forces which hold an antigen and antibody together are in essence nodifferent from non-specific interactions which occur between any twounrelated proteins i.e., other macromolecules such as human serumalbumin and human transferrin. These intermolecular forces may beclassified into four general areas which are (1) electrostatic; (2)hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostaticforces are due to the attraction between oppositely charged ionic groupson two protein side-chains. The force of attraction (F) is inverselyproportional to the square of the distance (d) between the charges.Hydrogen bonding forces are provided by the formation of reversiblehydrogen bridges between hydrophilic groups such as —OH, —NH₂ and —COOH.These forces are largely dependent upon close positioning of twomolecules carrying these groups. Hydrophobic forces operate in the sameway that oil droplets in water merge to form a single large drop.Accordingly, non-polar, hydrophobic groups such as the side-chains onvaline, leucine and phenylalanine tend to associate in an aqueousenvironment. Lastly, Van der Waals are forces created between moleculeswhich depend on interaction between the external electron clouds.

Further information regarding each of the different types of forces canbe obtained from “Essential Immunology” edited by I. M. Roitti (6thEdition) Blackwell Scientific Publications, 1988. with respect to thepresent invention useful antibodies exhibit all of these forces. It isby obtaining an accumulation of these forces in larger amounts that itis possible to obtain an antibody which has a high degree of affinity orbinding strength to the PrP protein and in particular an antibody whichhas a high degree of binding strength to PrP^(Sc) in situ.

Measuring Antibody/Anticen Binding Strength

The binding affinity between an antibody and an antigen can be measuredwhich measurement is an accumulation of a measurement of all of theforces described above. Standard procedures for carrying out suchmeasurements exist and can be directly applied to measure the affinityof antibodies of the invention for PrP proteins including nativePrP^(Sc) in situ.

One standard method for measuring antibody/antigen binding affinity isthrough the use of a dialysis sac which is a container comprised of amaterial which is permeable to the antigen but impermeable to theantibody. Antigens which are bound completely or partially to antibodiesare placed within the dialysis sac in a solvent such as in water. Thesac is then placed within a larger container which does not containantibodies or antigen but contains only the solvent e.g., the water.Since only the antigen can diffuse through the dialysis membrane theconcentration of the antigen within the dialysis sac and theconcentration of the antigen within the outer larger container willattempt to reach an equilibrium. After placing the dialysis sac into thelarger container and allowing for time to pass towards reaching anequilibrium it is possible to measure the concentration of the antigenwithin the dialysis sac and within the surrounding container and thendetermine the differences in concentration. This makes it possible tocalculate the amount of antigen which remains bound to antibody in thedialysis sac and the amount which disassociates from the antibody anddiffuses into the surrounding container. By constantly renewing thesolvent (e.g., the water) within the surrounding container so as toremove any antigen which is diffused thereinto it is possible to totallydisassociate the antibody from antigen within the dialysis sac. If thesurrounding solvent is not renewed the system will reach an equilibriumand it is possible to calculate the equilibrium constant (K) of thereaction i.e., the association and disassociation between the antibodyand antigen. The equilibrium constant (K) is calculated as an amountequal to the concentration of antibody bound to antigen within thedialysis sac divided by the concentration of free antibody combiningsites times the concentration of free antigen. The equilibrium constantor “K” value is generally measured in terms of liters per mole. The Kvalue is a measure of the difference in free energy (deta g) between theantigen and antibody in the free state as compared with the complexedform of the antigen and antibody. When using the phage displaymethodology described below the antibodies obtained have an affinity orK value of 10⁷ mole/liter or more.

Antibody Avidity

As indicated above the term “affinity” describes the binding of anantibody to a single antigen determinate. However, in most practicalcircumstances one is concerned with the interaction of an antibody witha multivalent antigen. The term “avidity” is used to express thisbinding. Factors which contribute to avidity are complex and include theheterogeneity of the antibodies in a given serum which are directedagainst each determinate on the antigen and the heterogeneity of thedeterminants themselves. The multivalence of most antigens leads to aninteresting “bonus” effect in which the binding of two antigen moleculesby an antibody is always greater, usually many fold greater, than thearithmetic sum of the individual antibody links. Thus, it can beunderstood that the measured avidity between an antiserum and amultivalent antigen will be somewhat greater than the affinity betweenan antibody and a single antigen determinate.

Null PrP Mice to make Antibodies

The present invention circumvents problems of tolerance and moreefficiently generates panels of monoclonal antibodies capable ofrecognizing diverse epitopes on Mo and other PrPs in part using micewith both alleles of the PrP gene (Prnp) are ablated (Prnp^(0/0))(Bueler, et al, 1992). These PrP-deficient mice (or null mice), areindistinguishable from normal mice in their development and behavior.These null mice are resistant to scrapie following intracerebralinoculation of infectious MoPrP^(Sc) (Bueler, et al, 1993 Cell;Prusiner, et al. PNAS 1993). In addition Prnp^(0/0) mice will developIgG serum titers against Mo-, SHa and human PrP following immunizationwith relatively small quantities of purified SHaPrP 27-30 in adjuvant(Prusiner et al. PNAS 1993). After allowing sufficient time to generateantibodies the immunized Prnp^(0/0) mice were sacrificed for hybridomaproduction in the conventional manner. Fusions derived from these micedid secrete PrP specific antibody. However, these hybridomas would notsecrete PrP specific antibodies for more than a few hours. In view ofthe somewhat limited success a different approach was taken.

Phage Display

Combinatorial antibody library technology, i.e., antigen based selectionfrom antibody libraries expressed on the surface of M13 filamentousphage, offers a new approach to the generation of monoclonal antibodiesand possesses a number of advantages relative to hybridoma methodologieswhich are particularly pertinent to the prion problem (Huse, et al,1989: Barbas, et al. 1991; Clackson, et al, 1991: Burton and Barbas,1994). The present invention uses such technology to providePrP-specific monoclonal antibodies from phage antibody librariesprepared from MoPrP-immunized Prnp^(0/0) mice. The invention providesthe first monoclonal antibodies recognizing MoPrP in situ anddemonstrates the application of combinatorial libraries for cloningspecific antibodies from null mice. The general methodologies involvedin creating large combinatorial libraries using phage display technologyis described and disclosed in U.S. Pat. No. 5,223,409 issued Jun. 29,1993 which patent is incorporated herein by reference to disclose anddescribe phage display methodology.

Null Animals

The invention is largely described herein with respect to null micei.e., FVB mice with both alleles of the PrP gene ablated. However, otherhost animals can be used and preferred host animals are mice andhamsters, with mice being most preferred in that there existsconsiderable knowledge on the production of transgenic animals. Possiblehost animals include those belonging to a genus selected from Mus (e.g.mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus(e.g. hamsters) and Cavia (e.g., guinea pigs). In general mammals with anormal full grown adult body weight of less than 1 kg which are easy tobreed and maintain can be used.

PrP Gene

The genetic material which makes up the PrP gene is known for a numberof different species of animals (see Gabriel et al., Proc. Natl. Acad.Sci. USA 89:9097-9101 (1992)). Further, there is considerable homologybetween the PrP genes in different mammals. For example, see the aminoacid sequence of mouse PrP compared to human, cow and sheep PrP in FIGS.2, 3 and 4 wherein only the differences are shown. Although there isconsiderable genetic homology with respect to PrP genes, the differencesare significant in some instances. More specifically, due to smalldifferences in the protein encoded by the PrP gene of different mammals,a prion which will infect one mammal (e.g. a human) will not normallyinfect a different mammal (e.g. a mouse). Due to this “species barrier”,it is not generally possible to use normal animals, (i.e., animal whichhave not had their genetic material related to PrP proteins manipulated)such as mice to determine whether a particular sample contains prionswhich would normally infect a different species of animal such as ahuman. The present invention solves this problem by providing antibodieswhich bind to native PrP^(Sc) proteins of any species of animal forwhich the antibody is designed.

Pathogenic Mutations and Polymorphisms

There are a number of known pathogenic mutations in the human PrP gene.Further, there are known polymorphisms in the human, sheep and bovinePrP genes. The following is a list of such mutations and polymorphisms:

Pathogenic human Human Sheep Bovine mutations PolymorphismsPolymorphisms Polymorphisms 2 octarepeat Codon 129 Codon 171 5 or 6insert Met/Val Arg/Glu octarepeats 4 octarepeat Codon 219 Codon 136insert Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala

The DNA sequence of the human, sheep and cow PrP genes have beendetermined allowing, in each case, the prediction of the complete aminoacid sequence of their respective PrP proteins. The normal amino acidsequence which occurs in the vast majority of individuals is referred toas the wild-type PrP sequence. This wild-type sequence is subject tocertain characteristic polymorphic variations. In the case of human PrP(SEQ ID NO:2), two polymorphic amino acids occur at residues 129(Met/Val) and 219 (Glu/Lys). Sheep PrP (SEQ ID NO:4) has two amino acidpolymorphisms at residues 171 and 136, while bovine PrP (SEQ ID NO:3)has either five or six repeats of an eight amino acid motif sequence inthe amino terminal region of the mature prion protein. While none ofthese polymorphisms are of themselves pathogenic, they appear toinfluence prion diseases. Distinct from these normal variations of thewild-type PrP proteins, certain mutations of the human PrP gene whichalter either specific amino acid residues of PrP or the number ofoctarepeats have been identified which segregate with inherited humanprion diseases.

In order to provide further meaning to the above chart demonstrating themutations and polymorphisms, one can refer to the published sequences ofPrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP geneare disclosed and published within Gabriel et al., Proc. Natl. Acad.Sci. USA 89:9097-9101 (1992). The sequence for the Syrian hamster ispublished in Basler et al., Cell 46:417-428 (1986). The PrP gene ofsheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA87:2476-2480 (1990). The PrP gene sequence for bovine is published inGoldmann et al., J. Gen. Virol. 72:201-204 (1991). The sequence forchicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci.USA 88:7664-7668 (1991). The PrP gene sequence for mink is published inKretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992). The human PrPgene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986).The PrP gene sequence for mouse is published in Locht et al., Proc.Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrP gene sequence forsheep is published in Westaway et al., Genes Dev. 8:959-969 (1994).These publications are all incorporated herein by reference to discloseand describe the PrP gene and PrP amino acid sequences.

“Strains” of Human Prions

Studies in rodents have shown that prion strains produce differentpatterns of PrP^(Sc) accumulation [Hecker et al., Genes & Development6:1213-1228 (1992); DeArmond et al., Proc. Natl. Acad. Sci. USA90:6449-6453 (1993)]; which can be dramatically changed by the sequenceof PrP^(Sc) [Carlson et al., Proc. Natl. Acad. Sci. USA in press(1994)]. The molecular basis of prion diversity has for many years beenattributed to a scrapie specific nucleic acid [Bruce et al., J. Gen.Virol. 68:79-89 (1987)] but none has been found [Meyer et al., J. Gen.Virol. 72:37-49 (1991); Kellings et al., J. Gen. Virol. 73:1025-1029(1992)]. Other hypotheses to explain prion strains include variations inPrP Asn-linked sugar chains [Hecker et al., Genes & Development6:1213-1228 (1992)] and multiple conformers of PrP^(Sc) [Prusiner, S.B., Science 252:1515-1522 (1991)]. The patterns of PrPSC in Tg(MHu2M)mice were remarkably similar for the three inocula from humans dying ofCJD.

The patterns of PrP^(Sc) accumulation in the brains of inoculatedTg(MHu2M) mice were markedly different for RML prions and Hu prions.However, RML prion inocula containing MoPrP^(Sc) stimulated theformation of more MoPrP^(Sc) while Hu prion inocula containingHuPrP^(CJD) triggered production of MHu2MPrP^(Sc). The distribution ofneuropathological changes characterized by neuronal vacuolation andastrocytic gliosis is similar to the patterns of PrP^(Sc) accumulationin the brains of Tg(MHu2M) mice inoculated with RML prions or Hu prions.

Standardized Prion Preparation

Standardized prion preparations may be produced in order to test assaysof the invention and thereby improve the reliability of the assay.Although the preparation can be obtained from any animal it ispreferably obtained from a host animal which has brain materialcontaining prions of a test animal. For example, a transgenic mousecontaining a human prion protein gene can produce human prions and thebrain of such a mouse can be used to create a standardized human prionpreparation. Further, in that the preparation is to be a “standard” itis preferably obtained from a battery (e.g., 100; 1,000, or moreanimals) of substantial identical animals. For example, 100 mice allcontaining a very high copy number of human PrP genes (all polymorphismsand mutations) would spontaneously develop disease and the brain tissuefrom each could be combined to make a useful standardized prionpreparation.

Standardized prion preparations can be produced using any of modifiedhost mammals of the type described above. For example, standardizedprion preparations could be produced using mice, rats, hamsters, orguinea pigs which are genetically modified so that they are susceptibleto infection with prions which prions would generally only infectgenetically diverse species such as a human, cow, sheep or horse andwhich modified host mammals will develop clinical signs of CNSdysfunction within a period of time of 350 days or less afterinoculation with prions. The most preferred host mammal is a mouse inpart because they are inexpensive to use and because a greater amount ofexperience has been obtained with respect to production of transgenicmice than with respect to the production of other types of host animals.Details regarding making standardized prion preparation are described inU.S. Patent application entitled “Method of Detecting Prions in a Sampleand Transgenic Animal Used For Same” filed Aug. 31, 1995, U.S. Ser. No.08/521,992 and U.S. Patent application entitled “Detecting Prions In ASample And Prion Preparation And Transgenic Animal Used For Same”,Attorney Docket No. 06510/056001, filed Jul. 30, 1996, both of whichapplications are incorporated herein by reference.

Once an appropriate type of host is chosen, such as a mouse, the nextstep is to choose the appropriate type of genetic manipulation to beutilized to produce a standardized prion formulation. For example, themice may be mice which are genetically modified by the insertion of achimeric gene of the invention. Within this group the mice might bemodified by including high copy numbers of the chimeric gene and/or bythe inclusion of multiple promoters in order to increase the level ofexpression of the chimeric gene. Alternatively, hybrid mice of theinvention could be used wherein mice which have the endogenous PrP geneablated are crossed with mice which have a human PrP gene inserted intotheir genome. There are, of course, various subcategories of such hybridmice. For example, the human PrP gene may be inserted in a high copynumber an/or used with multiple promoters to enhance expression. In yetanother alternative the mice could be produced by inserting multipledifferent PrP genes into the genome so as to create mice which aresusceptible to infection with a variety of different prions, i.e., whichgenerally infect two or more types of test animals. For example, a mousecould be created which included a chimeric gene including part of thesequence of a human, a separate chimeric gene which included part of thesequence of a cow and still another chimeric gene which included part ofthe sequence of a sheep. If all three different types of chimeric geneswere inserted into the genome of the mouse the mouse would besusceptible to infection with prions which generally only infect ahuman, cow and sheep.

After choosing the appropriate mammal (e.g., a mouse) and theappropriate mode of genetic modification (e.g., inserting a chimeric PrPgene) the next step is to produce a large number of such mammals whichare substantially identical in terms of genetic material related toprions. More specifically, each of the mice produced will include anidentical chimeric gene present in the genome in substantially the samecopy number. The mice should be sufficiently identical genetically interms of genetic material related to prions that 95% or more of the micewill develop clinical signs of CNS dysfunction within 350 days or lessafter inoculation and all of the mice will develop such CNS dysfunctionat approximately the same time e.g., within ±30 days of each other.

Once a large group e.g., 50 or more, more preferably 100 or more, stillmore preferably 500 or more of such mice are produced. The next step isto inoculate the mice with prions which generally only infect agenetically diverse mammal e.g., prions from a human, sheep, cow orhorse. The amounts given to different groups of mammals could be varied.After inoculating the mammals with the prions the mammals are observeduntil the mammals exhibit symptoms of prion infection e.g., clinicalsigns of CNS dysfunction. After exhibiting the symptoms of prioninfection the brain or at least a portion of the brain tissue of each ofthe mammals is extracted. The extracted brain tissue is homogenizedwhich provides the standardized prion preparation.

As an alternative to inoculating the group of transgenic mice withprions from a genetically diverse animal it is possible to produce micewhich spontaneously develop prion related diseases. This can be done,for example, by including extremely high copy numbers of a human PrPgene into a mouse genome. When the copy number is raised to, forexample, 100 or more copies, the mouse will spontaneously developclinical signs of CNS dysfunction and have, within its brain tissue,prions which are capable of infecting humans. The brains of theseanimals or portions of the brain tissue of these animals can beextracted and homogenized to produce a standardized prion preparation.

The standardized prion preparations can be used directly or can bediluted and tittered in a manner so as to provide for a variety ofdifferent positive controls. More specifically, various known amounts ofsuch standardized preparation can be used to inoculate a first set oftransgenic control mice. A second set of substantially identical miceare inoculated with a material to be tested i.e., a material which maycontain prions. A third group of substantially identical mice are notinjected with any material. The three groups are then observed. Thethird group, should, of course not become ill in that the mice are notinjected with any material. If such mice do become ill the assay is notaccurate probably due to the result of producing mice whichspontaneously develop disease. If the first group, injected with astandardized preparation, do not become ill the assay is also inaccurateprobably because the mice have not been correctly created so as tobecome ill when inoculated with prions which generally only infect agenetically diverse mammal. However, if the first group does become illand the third group does not become ill the assay can be presumed to beaccurate. Thus, if the second group does not become ill the testmaterial does not contain prions and if the second group does become illthe test material does contain prions.

By using standardized prion preparations of the invention it is possibleto create extremely dilute compositions containing the prions. Forexample, a composition containing one part per million or less or evenone part per billion or less can be created. Such a composition can beused to test the sensitivity of the antibodies, assays and methods ofthe invention in detecting the presence of prions.

Prion preparations are desirable in that they will include a constantamount of prions and are extracted from an isogeneic background.Accordingly, contaminates in the preparations will be constant andcontrollable. Standardized prion preparations will be useful in thecarrying out of bioassays in order to determine the presence, if any, ofprions in various pharmaceuticals, whole blood, blood fractions, foods,cosmetics, organs and in particular any material which is derived froman animal (living or dead) such as organs, blood and products thereofderived from living or dead humans. Thus, standardized prionpreparations will be valuable in validating purification protocols wherepreparations are spiked and reductions in teeter measured for aparticular process.

Useful Applications

As indicated above and described further below in detailed examples itis possible to use the methodology of the invention to create a widerange of different antibodies. i.e., antibodies having differentspecific features. For example, antibodies can be created which bindonly to a prion protein naturally occurring within a single species andnot bind to a prion protein naturally occurring within other species.Further, the antibody can be designed so as to bind only to aninfectious form of a prion protein (e.g., PrP^(Sc)) and not bind to anon-infectious form (e.g., PrP^(C)). A single antibody or a battery ofdifferent antibodies can then be used to create an assay device. Such anassay device can be prepared using conventional technology known tothose skilled in the art. The antibody can be purified and isolatedusing known techniques and bound to a support surface using knownprocedures. The resulting surface having antibody bound thereon can beused to assay a sample in vitro to determine if the sample contains oneor more types of antibodies. For example, antibodies which bind only tohuman PrP^(Sc) can be attached to the surface of a material and a samplecan be denatured via proteinase K. The denatured sample is brought intocontact with the antibodies bound to the surface of material. If nobinding occurs it can be deduced that the sample does not contain humanPrP^(Sc).

Antibodies of the invention are also characterized by their ability toneutralize prions. Specifically, when antibodies of the invention areallowed to bind to prions the infectivity of the prion is eliminated.Accordingly, antibody compositions of the invention can be added to anygiven product in order to neutralize any infectious prion protein withinthe product. Thus, if a product is produced from a natural source whichmight contain infectious prion proteins the antibodies of the inventioncould be added as a precaution thereby eliminating any potentialinfection resulting from infectious prion proteins.

The antibodies of the invention can be used in connection withimmunoaffinity chromatography technology. More specifically, theantibodies can be placed on the surface of a material within achromatography column. Thereafter, a composition to be purified can bepassed through the column. If the sample to be purified includes anyprion protein which binds to the antibodies those prion proteins(PrP^(Sc)) will be removed from the sample and thereby purified.

Lastly, the antibodies of the invention can be used to treat a mammal.The antibodies can be given prophylactically or be administered to anindividual already infected with infectious prion proteins suchinfection having been determined by the use of the assay describedabove. The exact amount of antibody to be administered will varydepending on a number of factors such as the age, sex, weight andcondition of the patient. Those skilled in the art can determine theprecise amount by administering antibodies in small amounts anddetermining the effect and thereafter adjusting the dosage. It issuggested that the dosage can vary from 0.01 mg/kg to about 300 mg/kg,preferably about 0.1 mg/kg to about 200 mg/kg, more preferably about 0.2mg/kg to about 20 mg/kg in one or more dose administrations daily, forone or several days. Preferred is administration of the antibody for 2to 5 or more consecutive days in order to avoid “rebound” of prioninfectivity occurring.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the chimeric genes, transgenic mice and assays of thepresent invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Construction of Phage Display Antibody libraries expressing antibodies(Fabs)

Construction of phage display libraries for expression of antibodies,particularly the Fab portion of antibodies, is well known in the art.Preferably, the phage display antibody libraries that express antibodiesare prepared according to the methods described in U.S. Pat. No.5,223,409, issued Jun. 29, 1993 and U.S. patent application Ser. No.07/945,515, filed Sep. 16, 1992, incorporated herein by reference.Procedures of the general methodology can be adapted using the presentdisclosure to produce antibodies of the present invention.

Isolation of RNA Encoding Prion-specific Antibodies

In general, the phage display anti-PrP antibody libraries are preparedby first isolating a pool of RNA that contains RNA encoding anti-PrPantibodies. To accomplish this, an animal (e.g., a mouse, rat, orhamster) is immunized with prion of interest. However, normal animals donot produce antibodies to prions at detectable or satisfactorily highlevels. This problem is avoided by immunizing animals in which the (PrP)gene has been ablated on both alleles. Such mice are designatedPrnp^(0/0) and methods for making such mice are disclosed in Büeler,Nature (1992) and in Weismann Publication WO 93/10227, published May 27,1993. Inoculation of “null” animals with prions results in production ofIgG serum titers against the prion (Prusiner et al. PNAS 1993). In onepreferred embodiment, the animal selected for immunization is aPrnp^(0/0) mouse described by Büeler and Weismann.

Generally, the amount of prion necessary to elicit a serum antibodyresponse in a “null” animal is from about 0.01 mg/kg to about 500 mg/kg.

The prion protein is generally administered to the animal by injection,preferably by intraperitoneal or intravenous injection, more preferablyby intraperitoneal injection. The animals are injected once, with atleast 1 to 4 subsequent booster injections, preferably at least 3booster injections. After immunization, the reactivity of the animal'santisera with the prion can be tested using standard immunologicalassays, such as ELISA or Western blot, according to methods well knownin the art (see, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Animals having prion-binding antisera may be boosted withan additional injection of prion.

Serum antibody levels are predictive of antibody secretion, andtherefore of levels of specific MRNA in lymphocytes, particularly plasmacells. Detection of serum antibodies, particularly relatively highlevels of serum antibodies, is thus correlated to a high level oflymphocytes such as plasma cells producing mRNA encoding those serumantibodies. Thus, plasma cells isolated from the prion protein-immunizedmice will contain a high proportion of lymphocytes (e.g., plasma cells)producing prion-specific antibody, particularly when the plasma cellsare isolated from the mice within a short time period after the finalinjection boost (e.g., about 2 to 5 days, preferably 3 days).Immunization of the mice and the subsequent injection boosters thusserve to increase the total percentage of anti-PrP antibody-producingplasma cells present in the total population of the mouse's plasmacells. Moreover, because the anti-PrP antibodies are being produced ator near peak serum levels, then anti-PrP antibody-producing plasma cellsare producing anti-PrP antibodies, and thus mRNA encoding theseantibodies at or near peak levels.

The above correlation between serum levels of antigen-specificantibodies, the number of lymphocytes producing those antigen-specificantibodies, and the amount of total mRNA encoding the antigen-specificantibodies provides a means for isolating a pool of MRNA that isenriched for the mRNA encoding antigen-specific antibodies of interest.Lymphocytes, including plasma cells are isolated from spleen and/or bonemarrow from the prion-immunized animals according to methods well knownin the art (see, for example, Huse et al. Science 1989). Preferably thelymphocytes are isolated about 2 to 5 days, preferably about 3 daysafter the final immunization boost. The total RNA is then extracted fromthese cells. Methods for RNA isolation from mammalian cells are wellknown in the art (see, for example, Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Production of CDNA Encoding Antibodies from Lymphocyte mRNA

cDNA is produced from the isolated RNA using reverse transcriptaseaccording to methods well known in the art (see, for example, Sambrooket al., supra), and CDNA encoding antibody heavy chains or light chainsis amplified using the polymerase chain reaction (PCR). The 3′ primersused to amplify heavy chain or light chain-encoding cDNAs are based uponthe known nucleotide sequences common to heavy chain or light chainantibodies of a specific antibody subclass. For example, one set ofprimers based upon the constant region of the IgG1 heavy chain-encodinggene can be used to amplify heavy chains of the IgG1 subclass, whileanother set of primers based upon the constant portion of the IgG1 lightchain-encoding gene is used to amplify the light chains of the IgG1subclass. The '5 primers are consensus sequences based upon examinationof a large number of variable sequences in the data base. In thismanner, DNA encoding all antibodies of a specific antibody class orsubclass are amplified regardless of antigen-specificity of theantibodies encoded by the amplified DNA. The entire gene encoding theheavy chain or the light chain can be amplified. Alternatively, only aportion of the heavy or light chain encoding gene may be amplified, withthe proviso that the product of PCR amplification encodes a heavy orlight chain gene product that can associate with its corresponding heavyor light chain and function in antigen binding i.e., bind selectively toa prion protein. Preferably, the phage display product is a Fab or Fvantibody fragment.

The antibody encoding cDNA selected for amplification may encode anyisotope and preferably encode a subclass of IgG. Exemplary mouse IgGsubclasses include IgG1, IgG2a, IgG2b, and IgG3. The selection of thespecific antibody subclass-encoding cDNA for amplification will varyaccording to a variety of factors, including, for example, the animal'sserum antibody response to the antigen. Preferably, the antibodysubclass-encoding cDNA selected for PCR amplification is that antibodysubclass for which the animal produced the highest titer of antibody.For example, if the titers of serum IgG1 are higher than any othersubclass of IgG detected in the serum antibody response, then cDNAencoding IgG1 is amplified from the cDNA pool.

Preferably, the heavy and light chains are amplified from the plasmacell cDNA to produce two separate amplified cDNA pools: 1) a cDNA poolcontaining heavy chain cDNA amplimer products, where the heavy chain isof a specific antibody subclass; and 2) a cDNA pool containing lightchain cDNA amplimer products, where the light chain is of a specificantibody subclass.

Antibodies From Transaenic Animals

In addition to obtaining genetic material which encodes antibodies byinfecting an animal with an antigen and thereafter extracting cells (andtheir DNA) responsible for antibody production it is possible to obtainthe genetic material by producing a transgenic animal or by using theabove described technology and transgenic animal technology in order toproduce chimeric mouse/human or fully human antibodies. The technologyfor producing a chimeric or wholly foreign immunoglobins involvesobtaining from cells of transgenic animals which have had inserted intotheir germ line a genetic material which encodes all or part of animmunoglobin which binds to the desired antigen. Wholly human antibodiescan be produced from transgenic mice which have had inserted into theirgenome genetic material which encodes human antibodies. The technologyfor producing such antibodies from transgenic animals is describedwithin PCT Publication No. WO 90/04036, published Apr. 19, 1990.Further, see Goodhartd, et al, Proc. Natl. Acad. Sci. U.S.A. Vol. 84,pages 4229-4233, June 1987 and Bucchine, et al, Nature, Vol. 326, pages409-411, Mar. 26, 1987, all of which are incorporated herein byreference to disclose and describe methods of producing antibodies fromtransgenic animals.

Vectors for Use with Phage Display Antibody Libraries

The heavy chain-encoding cDNAs and the light chain-encoding cDNAs arethen each inserted into separate expression cassettes of an appropriatevector. Preferably the vector contains a nucleotide sequence encodingand capable of expressing a fusion polypeptide containing, in thedirection of amino- to carboxy-terminus, 1) a prokaryotic secretionsignal domain, 2) an insertion site for DNA encoding a heterologouspolypeptide (e.g., either the heavy or light chain-encoding cDNA), andin the expression cassette for the heavy chain cDNA 3) a filamentousphage membrane anchor domain.

The vector includes prokaryotic or mammalian DNA expression controlsequences for expressing the fusion polypeptide, preferably prokaryoticcontrol sequences. The DNA expression control sequences can include anyexpression signal for expressing a structural gene product, and caninclude 5′ and 3′ elements operatively linked to the expression cassettefor expression of the heterologous polypeptide. The 5′ control sequencedefines a promoter for initiating transcription, and a ribosome bindingsite operatively linked at the 5′ terminus of the upstream translatablesequence. The vector additionally includes an origin of replication formaintenance and replication in a prokaryotic cell, preferably a gramnegative cell such as E. coli. The vector can also include genes whoseexpression confers a selective advantage, such as drug resistance, to aprokaryotic or eukaryotic cell transformed with the vector.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface. The secretion signal is a leader peptide domainof a protein that targets the protein to the periplasmic membrane ofgram negative bacteria. Such leader sequences for gram negative bacteria(such as E. coli) are well known in the art (see, for example, Oliver,In Neidhard, F. C. (ed.), Escherichia coli and Salmonella typhimurium,American Society for Microbiology, Washington, D.C., 1:56-69, 1987).

Filamentous Phage Membrane Anchors for use in the Phage Display Vector

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged aminoacid residues normally found at the cytoplasmic face of the membrane andextending away from the membrane. In the page f1, gene VIII coatprotein's membrane spanning region comprises the carboxy-terminal 11residues from 41 to 52 (Ohkawa et al., J. Biol. Chem., 256:9951-9958,1981). An exemplary membrane anchor would consist of residues 26 to 40to cpVIII. Thus, the amino acid residue sequence of a preferred membraneanchor domain is derived from the M13 filamentous phage gene VIII coatprotein (also designated cpVIII or CP 8). Gene VIII coat protein ispresent on a mature filamentous phage over the majority of the phageparticle with typically about 2500 to 3000 copies of the coat protein.

The amino acid residue sequence of another preferred membrane anchordomain is derived from the M13 filamentous phage gene III coat protein(also designate cpIII). Gene III coat protein is present on a maturefilamentous phage at one end of the phage particle with typically about4 to 6 copies of the coat protein. Detailed descriptions of thestructure of filamentous phage particles, their coat proteins, andparticles assembly are found in the reviews by Rached et al.,(Microbiol. Rev., 50:401-427, 1986) and Model et al. (In: TheBacteriophages: Vol. 2, R. Calendar, ed., Plenum Publishing Co., pgs.375-456, 1988).

Preferably, the filamentous phage membrane anchor-encoding DNA isinserted 3′ of the cDNA insert in the library vector such that the phagemembrane anchor-encoding DNA can be easily excised and the vectorrelegated without disrupting the rest of the expression cassettes of thevector. Removal of the phage membrane anchor-encoding DNA from thevector, and expression of this vector in an appropriate host cell,results in the production of soluble antibody (Fab) fragments. Thesoluble Fab fragments retain the antigenicity of the phage-bound Fab,and thus can be used in assays and therapies in the manner that whole(non-fragmented) antibodies are used.

The vector for use with the present invention must be capable ofexpressing a heterodimeric receptor (such as an antibody or antibodyFab). That is, the vector must be capable of independently containingand expressing two separate cDNA inserts (e.g., the heavy chain cDNA andthe light chain cDNA). Each expression cassette can include the elementsdescribed above, except that the filamentous phage anchormembrane-encoding DNA is present only in the expression cassette for theheavy chain cDNA. Thus, when the antibody or Fab is expressed on thesurface of the phage, only the heavy chain polypeptide is anchored tothe phage surface. The light chain is not directly bound to the phagesurface, but is indirectly bound to the phage via its association withthe free portion of the heavy chain polypeptide (i.e., the portion ofthe heavy chain that is not bound to the phage surface).

Preferably, the vector contains a sequence of nucleotides that allow fordirectional ligation, i.e., a polylinker. The polylinker is a region ofthe DNA expression vector that operatively links the upstream anddownstream translatable DNA sequence for replication and transport, andprovides a site or means for directional ligation of a DNA sequence intothe vector. Typically, a directional polylinker is a sequence ofnucleotides that defines two or more restriction endonucleaserecognition sequence, or restriction sites. Upon restriction enzymecleavage, the two sites yield cohesive termini to which a translatableDNA sequence can be ligated to the DNA expression vector. Preferably,the two cohesive termini are non-complementary and thereby permitdirectional insertion of the cDNA into the cassette. Polylinkers canprovide one or multiple directional cloning sites, and may or may not betranslated during expression of the inserted cDNA.

Preferably, the expression vector is capable of manipulating in the formof a filamentous phage particle. Such DNA expression vectorsadditionally contain a nucleotide sequence that defines a filamentousphage origin of replication such that the vector, upon presentation ofthe appropriate genetic complement, can replicate as a filamentous phagein single stranded replicative form, and can be packaged intofilamentous phage particles. This feature provides the ability of theDNA expression vector to be packaged into phage particles for subsequentisolation of individual phage particles (e.g., by infection of andreplication in isolated bacterial colonies).

A filamentous phage origin of replication is a region of the phagegenome that defines sites for initiation of replication, termination ofreplication, and packaging of the replicative form produced byreplications (see, for example, Rasched et al., Microbiol. Rev.,50:401-427, 1986; Horiuchi, J. Mol. Biol., 188:215-223, 1986). Apreferred filamentous phage origin of replication for use in the presentinvention is an M13, f1, or fd phage origin of replication (Short etal., Nucl. Acids Res., 16:7583-7600, 1988). Preferred DNA expressionvectors are the expression vectors pCOMB8, pCKAB8, pCOMB2-8, pCOMB3,pCKAB3, pCOMB2-3, pCOMB2-3′ and pCOMB3H.

The pComb3H vector is a modified form of pComb3 in which (i) heavy andlight chains are expressed from a single Lac promoter as opposed toindividual promoters and (ii) heavy and light chains have two differentleader sequences (pg1B and ompA) as opposed to the same leader sequence(pH2). Reference for pComb3H Wang, et al (1995) J. Mol. Biol., Inpress.The principles of pComb3H are basically the same as for pComb3.

Production of the Phage Display Antibody Library

After the heavy chain and light chain cDNAs are cloned into theexpression vector, the entire library is packaged using an appropriatefilamentous phage. The phage are then used to infect a phage-susceptiblebacterial culture (such as a strain of E. coli), the phage allowed toreplicate and lyse the cells, and the lysate isolated from the bacterialcell debris. The phage lysate contains the filamentous phage expressingon its surface the cloned heavy and light chains isolated from theimmunized animal. In general, the heavy and light chains are present onthe phage surface as Fab antibody fragments, with the heavy chain of theFab being anchored. to the phage surface via the filamentous phagemembrane anchor portion of the fusion polypeptide. The light chain isassociated with the heavy chain so as to form an antigen binding site.Method of producing chimeric antibodies are described within U.S. Pat.No. 4,816,567, issued Mar. 28, 1989 to Cabilly, et al which isincorporated herein by reference to disclose and describe suchprocedures. Further, See Bobrzecka, et al, Immunology Letters, 2, pages151-155 (1980) and Konieczny, et al, Haematologia 14 (1), pages 85-91(1981) also incorporated herein by reference.

Selection of Prion-antigen Specific Fabs from the Phage Display AntibodyLibrary

Phage expressing an antibody or Fab that specifically binds a prionantigen can be isolated using any of a variety of protocols foridentification and isolation of monoclonal and/or polyclonal antibodies.Such methods include, immunoaffinity purification (e.g., binding of thephage to a column a having bound antigen) and antibody panning methods(e.g., repeated rounds of phage binding to antigen bound to a solidsupport for selection of phage of high binding affinity to the antigen).Preferably, the phage is selected by panning using techniques that arewell known in the art.

After identification and isolation of phage expressing anti-PrPantibodies, the phage can be used to infect a bacterial culture, andsingle phage isolates identified. Each separate phage isolate can beagain screened using one or more of the methods described above. Inorder to further confirm the affinity of the phage for the antigen,and/or to determine the relative affinities of the phage for theantigen, the DNA encoding the antibodies or Fabs can be isolated fromthe phage, and the nucleotide sequence of the heavy and light chainscontained in the vector determined using methods well known in the art(see, for example, Sambrook et al., supra).

Isolation of Soluble Fabs from Phage Selected from the Phage DisplayAntibody Library

Soluble antibodies or Fabs can be produced from a modified display thesame dicistronic vector by excising the DNA encoding the filamentousphage anchor membrane that is associated with the expression cassettefor the heavy chain of the antibody. Preferably, the DNA encoding theanchor membrane is flanked by convenient restriction sites that allowexcision of the anchor membrane sequence without disruption of theremainder of the heavy chain expression cassette or disruption of anyother portion of the expression vector. The modified vector without theanchor membrane sequence then allows for production of soluble heavychain as well as soluble light chain following packaging and infectionof bacterial cells with the modified vector.

Alternatively, where the vector contains the appropriate mammalianexpression sequences the modified vector can be used to transform aeukaryotic cell (e.g., a mammalian or yeast cell, preferably a mammaliancell (e.g., Chinese hamster ovary (CHO) cells)) for expression of theFab. Where the modified vector does not provide for eukaryoticexpression, preferably the vector allows for excision of both the heavyand light chain expression cassettes as a single DNA fragments forsubcloning into an appropriate vector. Numerous vectors for expressionof proteins in prokaryotic and/or eukaryotic cells are commerciallyavailable and/or well known in the art (see, for example Sambrook etal., supra).

Commercial Assay

Examples 14-18 below and specifically Example 17 show the isolation ofan antibody which specifically binds to PrP^(Sc) without anydenaturation. A sample containing PrP proteins (i.e., PrP^(C) andPrP^(Sc)) can be subjected to denaturation by the use of protease K (PK)digestion. The use of such will digest PrP^(C) but not PrP^(Sc). Thus,after carrying out the digestion the sample is contacted with theantibody (e.g., R2) as per Example 17 under suitable binding conditions.Preferably, the antibody is bound to a substrate and can be positionedsuch that the sample can be easily contacted with the substrate materialhaving the antibody bound thereon. If material binds to the antibodieson the substrate the presence of infectious PrP^(Sc) is confirmed.

In commercial embodiments of the invention it may be desirable to useantibodies of the invention in a sandwich type assay. More particularly,the antibody of the invention may be bound to a substrate supportsurface. The sample to be tested is contacted with the support surfaceunder conditions which allow for binding. Thereafter, unreacted sitesare blocked and the surface is contacted with a generalized antibodywhich will bind to any protein thereon. The generalized antibody islinked to a detectable label. The generalized antibody with detectablelabel is allowed to bind to any PrP^(Sc)bound to the antibodies on thesupport surface. If binding occurs the label can be made to becomedetectable such as by generating a color thereby indicating the presenceof the label which indirectly indicates the presence of PrP^(Sc) withinthe sample. The assay can detect prions (PrP^(Sc)) present in an amountof 1 part per million or less, even one part per billion or less. ThePrP^(Sc) may be present in a source selected from the group consistingof (a) a pharmaceutical formulation containing a therapeutically activecomponent extracted from an animal source, (b) a component extractedfrom a human source, (c) an organ, tissue, body fluid or cells extractedfrom a human source, (d) a formulation selected form the groupconsisting of injectables, orals, creams, suppositories, andintrapulmonary delivery formulations, (e) a cosmetic, and (f) apharmaceutically active compound extracted from a mammalian cellculture. Such source materials can also be treated to remove orneutralize PrP^(Sc) protein by adding an antibody of the invention. Theinvention also includes a method of treating, comprising administeringto a mammal in need thereof a therapeutically effective amount of anantibody which selectively binds PrP^(Sc) protein which antibody ischaracterized by its ability to neutralize PrP^(Sc) protein infectivity.

Generalized Procedure

Antibodies of the invention could be obtained by a variety oftechniques. However, the general procedure involves synthesizing alibrary of proteins (i.e., antibodies or portions thereof) on thesurface of phage. The library is then brought into contact with acomposition which includes PrP proteins and in particular is a naturallyoccurring composition which includes PrP^(Sc). The phage which bind toPrP protein are then isolated and the antibody or portion thereof whichbinds the PrP protein is isolated. It is desirable to determine thesequence of the genetic material encoding the antibody or portionthereof. Further, the sequence can be amplified and inserted, by itself,or with other genetic material into an appropriate vector and cell linefor the production of other antibodies. For example, a sequence encodinga variable region which binds PrP^(Sc) can be fused with a sequencewhich encodes a human constant region of an antibody producing aconstant/variable construct. This construct can be amplified andinserted within a suitable vector which can be inserted within asuitable cell line for the production of humanized antibodies.Procedures such as this are described within U.S. Pat. No. 4,816,567,issued Mar. 28, 1989 to Cabilly, et al which is incorporated herein byreference to disclose and describe such procedures. Further, SeeBobrzecka, et al, Immunology Letters, 2, pages 151-155 (1980) andKonieczny, et al, Haematologia 14 (1), pages 85-91 (1981) alsoincorporated herein by reference.

When the genetic material encoding an antibody or portion thereof whichbinds a PrP protein is isolated it is possible to use that geneticmaterial to produce other antibodies or portions thereof which have agreater affinity for binding PrP proteins. This is done by site directedmutagenesis technology or by random mutagenesis and selection.Specifically, individual codons or groups of codons within the sequenceare removed or replaced with codons which encode different amino acids.Large numbers of different sequences can be generated, amplified andused to express variations of the antibody or portions thereof on thesurface of additional phage. These phage can then be used to test forthe binding affinity of the antibody to PrP proteins.

The phage library can be created in a variety of different ways. Inaccordance with one procedure a host animal such as a mouse or rat isimmunized with PrP protein and preferably immunized with PrP^(Sc). Theimmunization may be carried out along with an adjuvant for the formationof larger amounts and types of antibodies. After allowing for sufficienttime for the generation of antibodies, cells responsible for antibodyproduction are extracted from the inoculated host mammal. RNA isisolated from the extracted cells and subjected to reverse transcriptionin order to produce a cDNA library. The extracted cDNA is amplified bythe use of primers and inserted into an appropriate phage displayvector. The vector allows the expression of antibodies or portionsthereof on the phage surface. It is also possible to subject the cDNA tosite directed mutagenesis prior to insertion into the display vector.Specifically, codons are removed or replaced with codons expressingdifferent amino acids in order to create a larger library (i.e., alibrary of many variants) which is then expressed on the surface of thephage. Thereafter, as described above, the phage are brought intocontact with the sample and phage which bind to PrP protein areisolated.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the recombinant anti-PrP antibodies and assays of thepresent invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1

Purification of MoPrP 27-30

Purified MoPrP 27-30 rods were prepared from the brains of clinicallyill CD-1 mice inoculated with RML prions (Chandler scrapie isolate(Chandler R. L. 1961 Lancet, 1378-1379)). Prion rods were recovered fromsucrose gradient fractions as previously described (Prusiner, McKinley1983 Cell). Briefly, the fractions containing prion rods, which sedimentin 48-60% (wt/vol) sucrose, were diluted 2:1 in distilled water andcentrifuged at 100,000×g for 6 h at 4° C. The pellet was resuspended inwater, centrifuged again, and the rods resuspended at 1 mg/ml inCa/Mg-free phosphate buffered saline (PBS) containing 0.2% Sarcosyl. PrP27-30 was the major protein as determined by SDS-PAGE and silverstaining analysis. Protein quantitation was performed by bicinchonicacid dye binding, with a known amount of bovine serum albumin as theprotein concentration standard.

Example 2

Immunization of Prnp^(0/0) Mice

Prnp^(0/0) mice, in which both alleles of the PrP gene (Prnp) isablated, were immunized with the purified MoPrP 27-30 rods, which wereisolated as described in Example 1. Prnp^(0/0) mice and methods formaking this strain are well known in the art (Büeler, et al. 1992).Prnp^(0/0) mice, which are indistinguishable from normal mice in theirdevelopment and behavior, are resistant to scrapie followingintracerebral inoculation of infectious MoPrP^(Sc)(Büeler, et al. 1993Cell; Prusiner et al. PNAS 1993), and will develop IgG serum titersagainst Mo-, SHa, and human PrP following immunization with relativelysmall quantities of purified SHaPrP 27-30 in adjuvant (Prusiner et al.PNAS 1993).

Three (3) six week old Prnp^(0/0) mice were immunized by intraperitonealinjection of 100 μg of MoPrP 27-30 rods fully emulsified in completeFreund's adjuvant. Subsequently mice were boosted 2 times at 2-weekintervals with incomplete Freund's adjuvant containing in the firstinstance 100 μg, then 50 μg of rods. Four days after the second boost,the reactivity of each mouse's serum against prion proteins was analyzedas described below in Example 3. Those mice having anti-PrP reactiveantisera received a third injection boost of 50 μg prion rods inincomplete Freund's adjuvant 14 days after the second boost.

Example 3

Serum Reactivity of Prnp^(0/0) Mice Immunized with MoPrP 27-30

A primary prognostic indicator for success in isolating a specificantibody from combinatorial libraries is serum antibody reactivity withthe antigen(s) to be studied (Burton and Barbas, Adv. Immunol. 1994).Serum antibody levels are predictive of antibody secretion and thereforepredictive of the levels of specific MRNA in plasma cells. It is thislatter factor that ultimately dictates the composition of theantibody-encoding cDNA library.

Four days after the second boost, the Prnp^(0/0) mice immunized withMoPrP 27-30 as described in Example 2 were bled from the tail, and theantisera stored at −20° C. for subsequent immunological analysis. Thereactivity of the immunized mouse serum (IgG1, IgG2a, IgG2b and IgG3antibody subclasses) was measured against denatured and non-denaturedMo- and SHaPrP 27-30 in ELISA. ELISA wells were coated overnight at 4°C. with 50 μl of PrP rods at 40 μg/ml in 100 mM sodium bicarbonate pH8.6. Where denatured PrP rods were used as the antigen in the ELISA, 50μl of 6M guanidinium isothiocyanate was added to the well for 15 min atroom temperature, after which the wells were washed 6 times withCa/Mg-free PBS. All wells were then blocked with Ca/Mg-free PBScontaining 30 BSA. The antisera was serially diluted in PBS, andincubated with the wells for one hour at 37° C. Excess antisera wasremoved by washing 10 times with PBS 10.05% Tween 20 and bound antiseradetected using labeled goat anti-mouse antibody that specifically bindseither IgG1, IgG2a, IgG2b or IgG3 murine antibodies.

All 3 mice produced anti-PrP IgG antibodies. Serum reactivity from oneof the mice, designated D7282, is illustrated in FIG. 5 as exemplary ofthe antibody responses of the immunized mice. The highest serum titersagainst Mo- and SHaPrP antigens were of the IgG1 and IgG2b subclasses.In contrast, the IgG2a and IgG3 anti-PrP titers were close to thebackground levels of reactivity seen for all IgG subclasses in the serumof non-immunized Prnp^(0/0) mice. Antibody titers were greater againstdenatured rods than non-denatured rods. The similar serum reactivityagainst Mo- and SHa denatured rods is likely reflective of the highamino acid sequence homology between the two proteins. However, althoughthere was considerable serum reactivity against non-denatured Mo- rods(approximately 40-50% of the level of that for denatured MoPrP 27-30),reactivity with non-denatured SHa rods was at the level of background.

Example 4

Isolation of mRNA Encoding Anti-PrP Antibodies and Construction ofAntibody Phage Display Libraries

Three days after the final injection boost, the D7282 mouse wassacrificed and RNA prepared from bone marrow and splenic tissues. TotalRNA from mouse spleen was prepared according to methods well known inthe art (Huse, et al Science 1989). RNA was prepared from bone marrowtissues by first removing the tibia and fibula from both rear legs ofthe mice. The bones were then cut through close to each end, and theircontents flushed out by injection of guanidinium isothiocyanate into thebone cavity using a 27 gauge needle. RNA preparation was then continuedas described for the mouse spleen.

The RNA preparations were then pooled, and cDNA generated from the MRNAusing reverse transcriptase according to methods well known in the art.Two cDNA libraries were independently constructed from the D7282 mousemRNA: 1) an IgG1 library; and 2) a IgG2b library. For each of theselibraries, cDNAs encoding heavy chains and cDNA light chains wereseparately amplified by PCR from separate fractions of the pooled cDNA.The oligonucleotide 5′ and 3′ primers employed for PCR amplification ofDNA fragments encoding murine light (K) chains and heavy (α1 or α2b)chains of the IgG1 subclass wee those used by Huse, et al (Science 1989)and additional heavy chain primers as presented in Table 1 and heavychain polymers which are presented in Table 1. Primers used foramplification of cDNAs encoding heavy chain fragments.

TABLE 1 HEAVY CHAIN PRIMERS Primer Nucleotide Sequence MVH 1b (SEQ IDNOS:5,6) 5′-[CG]AG GTG CAG CTC GAG GAG TCA GGA CCT-3′ MVH 2b (SEQ IDNO:7) 5′-GAG GTC CAG CTC GAG CAG TCT GGA CCT-3′ MVH 3b (SEQ ID NO:8)5′-CAG GTC CAA CTC GAG CAG CCT GGG GTC-3′ MVH 4b (SEQ ID NO:9) 5′-GAGGTT CAG CTC GAG CAG TCT GGG GCAA-3′ MVH 5b (SEQ ID NOS:10,11)5′-GA[AG]GTG AAG CTC GAG GAG TCT GGA GGA-3′ MVH 6b (SEQ ID NO:12) 5′-GAGGTG AAG CTT CTC GAG TCT GGA GGT-3′ MVH 7b (SEQ ID NO:13) 5′-GAA GTG AAGCTC GAG GAG TCT GGG GGA-3′ MVH 8b (SEQ ID NO:14) 5′-GAG GTT CAG CTC GAGGAG CAG TCT GGA GCT-3′ MVH 1a (SEQ ID NOS:15-46) 5′-AGG T[CG][CA] A[GA]CT[GT]C TCG AGT C[TA]GG-3′ MVH 2a (SEQ ID NO:47) 5′-AGG TCC AGC TGC TCGAGT CTG G-3′ MVH 3a (SEQ ID NO:48) 5′-AGG TCC AGC TGC TCG AGT CAG G-3′MVH 4a (SEQ ID NO:49) 5′-AGG TCC AGC TTC TCG AGT CTG G-3′ MVH 5a (SEQ IDNO:50) 5′-AGG TCC AGC TTC TCG AGT CAG G-3′ Primers used for theAmplification of Antibody Light Chain Fragments 5′ PRIMERS MVK 1 (SEQ IDNO:51) 5′-CCA GTT CCG AGC TCG TTG TGA CTC AGG AAT CT-3′ MVK 2 (SEQ IDNO:52) 5′-CCA GTT CCG AGC TCG TGG TGA CGC AGC CGC CC-3′ MVK 3 (SEQ IDNO:53) 5′-CCA GTT CCG AGC TCG TGC TCA CCC AGT CTC CA-3′ MVK 4 (SEQ IDNO:54) 5′-CCA GTT CCG AGC TCC AGA TGA CCC AGT CTC CA-3′ MVK 5 (SEQ IDNO:54) 5′-CCA GAT GTG AGC TCG TGA CCC AGA CTC CA-3′ MVK 6 (SEQ ID NO:55)5′-CCA GAT GTG AGC TCG TCA TGA CCC AGT CTC CA-3′ MVK 7 (SEQ ID NO:56)5′-CCA GTT CCG AGC TCG TGA TGA CAC AGT CTC CA-3′ 3′ PRIMERS MCK 1 (SEQID NO:58) 5′-GCG CCG TCT AGA ATT AAC ACT CAT TCC TGT TGA A-3′ MVH 6a(SEQ ID NO:59) 5′-AGG TCC AAC TGC TCG AGT CTG G-3′ MVH 7a (SEQ ID NO:60)5′-AGG TCC AAC TGC TCG AGT TCA G-3′ MVH 8a (SEQ ID NO:61) 5′-AGG TCC AACTTC TCG AGT CTG G-3′ 3′ PRIMERS MIgGI (SEQ ID NO:62) 5′-AGG CTT ACT AGTACA ATC CCT GGG CAC AAT-3′ MIgG2B (SEQ ID NO:63) 5′-CTC CTT ACT AGT AGGACA GGG GAT TGT-3′

PCR was performed using a Perkin Elmer 9600 with 35 rounds ofamplification; denaturation at 94° C. for 30 sec, hybridization at 52°C. for 60 sec and extension at 72° C. for 60 sec.

The resulting amplified cDNAs encoding heavy chains of the IgG1 andIgG2b subclasses and light chains were cloned into the vector pComb3.The preparation of Fab antibody libraries displayed on the surface of afilamentous phage using the pComb3 vector have been described(Williamson et al. PNAS, 1993; Barbas et al. PNAS 1991). Briefly, theIgG1 or IgG2b phage display library is constructed by inserting theamplified CDNA encoding IgG1 or IgG2b heavy chain and the amplified cDNAencoding light chain into the pComb3H vector such that each vectorcontains a cDNA insert encoding a heavy chain fragment in one expressioncassette of the vector, and a cDNA insert encoding a light chainfragment into the other expression cassette of the vector. The resultingIgG1 library contained approximately 9×10⁶ individual clones, while theresulting IgG2b library contained approximately 7×10⁶ individual clones.

The ligated vectors were then packaged by the filamentous phage M13using methods well known in the art (see, for example, Sambrook et al,supra). The packaged library is then used to infect a culture of E.coli, so as to amplify the number of phage particles. After bacterialcell lysis, the phage particles are isolated and used in the panningprocedure that follows. Aliquots of the phage library are stored forfuture amplification and use. Separate aliquots of the phage librariesare isolated and stored for future amplification and use.

Example 5

Screening of the Phage Display Antibody Library for Binding to PrP

Antigen binding phage were selected for binding to denatured MoPrP 27-30rods against PrP antigen bound to ELISA wells through a panningprocedure described in (Burton, et al PNAS 1991, Barbas Lerner Methodsin Enzymol 1991). Briefly, ELISA wells were coated overnight at 4° C.with 50 μl of MoPrP 27-30 rods at 40 μg/ml in 100 mM sodium bicarbonatepH 8.6. The PrP rods were then denatured by incubation with 50 μl of 6Mguanidinium isothiocyanate for 15 min at room temperature, after whichthe wells were washed 6 times with Ca/Mg-free PBS. The wells were thenblocked with Ca/Mg-free PBS containing 3% BSA.

Aliquots of antibody phage were applied to separate PrP coated ELISAwells. A total of approximately 1×10¹⁰ antibody phage were added perwell in the panning experiment.

The phage were incubated with the well-bound MoPrP antigen for 2 hrs at37° C. Unbound phage were removed by washing 10 times with PBS 0.5%TWEEN 20. Bound phage were then removed from the wells by acid elution,pooled, reamplified and subjected to a second round of panning.

The IgG1 library was selected through 5 rounds of panning. A 40-foldamplification of PrP-specific antibody phage, as determined by thenumber of phage eluted from PrP-coated ELISA wells, was measured fromthe first to the fifth round.

Example 6

Soluble Fab Production from Selected Antibody-producing Phage

Soluble Fabs were produced from phage clones eluted from the fourth andfifth rounds of panning. DNA from the selected phage clones wasisolated, and the phage coat protein III (the filamentous phage membraneanchor) was removed from the pComb3H vector using the appropriaterestriction enzymes. The DNA was self-ligated to yield a vector capableof expressing soluble Fab (the procedure for production of soluble Fabsis detailed in (Barbas et al. PNAS 1991)). The vectors were thenseparately used to transform bacteria for expression of the Fabs, andisolated transformants were selected.

Fab expression was induced in an overnight bacterial culture usingisopropyl β-D-thiogalactopyranoside. The bacteria were centrifuged, andthe resulting bacterial pellet was either sonicated or frozen and thawedthree times to release Fab from the bacterial periplasmic space. Thebacterial Fab supernatants were then tested for reactivity against PrPin ELISA.

Example 7

ELISA Analysis of Anti-PrP Fabs Binding to PrP Antigens

The binding of soluble Fabs produced in Example 6 to denatured andnon-denatured PrP antigens as well as to synthetic PrP peptides wasdetermined using the ELISA assay described in Example 3. Synthetic PrPpeptides were produced using conventional peptide synthesis protocolswell known in the art.

Of the Fab clones taken from the fourth round of the panning againstdenatured MoPrP rods, less than 5% were reactive with denatured PrP,while approximately 50% of the clones taken from the fifth round of thesame panning recognized PrP antigens. In ELISA all of the reactiveclones from this panning were able to bind specifically to denatured Moand SHa rods, but not to non-denatured rods from either species. Inaddition, all the anti-PrP Fabs failed to recognize synthetic peptidesspanning residues 90-145 of Mo and SHa PrP, suggesting the antibodiesbind between residues 146 and 231 of the prion protein.

Example 8

Analysis of Selected Anti-PrP Antibody (Fab) Binding to Prion-infectedand Uninfected Rodent Brain Tissue

The reactivity of the antibodies identified by panning of the phagedisplay antibody library was tested by SDS/PAGE of prion-infected rodentbrain tissue and Western blot analysis using the selected Fabs. Proteinfrom brain tissues of prion-infected and uninfected mice was used as theantigen against which immunoreactivity was tested. The antigen wasprepared by disrupting rodent brain tissue in Ca/Mg-free PBS by passage5 times through a 20 gauge needle, followed by passage 10 times througha 22 gauge needle. The 10% (wt/vol) homogenate was then centrifuged at1600×g for 5 min at 4° C. Aliquots of the supernatant protein werediluted to a final concentration of 1 mg/ml in Ca/Mg-free PBS containing0.2% Sarcosyl. This dilution was mixed with an equal volume ofnon-reducing 2×SDS/PAGE sample buffer and boiled for 5 min, beforeSDS/PAGE (Laemmli. U.K. (1970) Nature (London) 227, 680-685).Immunoblotting was performed as previously described (Pan et al, PNAS1993) with primary mouse IgG antiserum (Pierce) diluted 1:1000.

Example 9

Nucleic Acid Sequencing

The nucleotide and amino acid sequences of the variable domains of theantibody light and heavy chains were determined for several of the PrPspecific clones. Nucleic acid sequencing was performed with a model 373Aautomated DNA sequencer (Applied Biosystems) using a Taq fluorescentdideoxynucleotide terminator cycle sequencing kit (Applied Biosystems).Primers for the elucidation of antibody light-chain sequence wereprimers MoSeqKb (SEQ ID NO:64) [5′-CAC GAC TGA GGC ACC TCC-3′] andOmpSeq (SEQ ID NO:65) [5′-AAG ACA GCT ATC GCG ATT GCA G-3′] hybridizingto the (−)-strand and for the heavy chain MOIgGGzSeq (SEQ ID NO:66)[5′-ATA GCC CTT GAC CAG GCA TCC CAG GGT CAC-3′] binding to the(+)-strand and PelSeq (SEQ ID NO:67) [5′-ACC TAT TGC CTA CGG CAG CCG-3′]binding to the (−)-strand.

The deduced amino acid sequences for some of the phage clones obtainedin one panning against denatured PrP are provided in FIGS. 6 (SEQ IDNOS:68-74) and 7(SEQ ID NOS:75-86). FIG. 6 shows the amino acidsequences of selected (A) heavy chain and (B) light chain variableregions generated by panning an IgG1 library from mouse D7282 againstdenatured MoPrP 27-30 rods. The sequences are very similar but contain anumber of heterogeneities which are likely the result of somaticmutation following repeated exposure of the mouse to PrP antigen. All ofthe heavy chain sequences examined in these clones contained verysimilar sequences. In particular, the heavy chain complementaritydetermining region 3(HCDR3) was identical at the nucleotide level in allthe Fab clones examined. Small differences were observed in the CDR1,CDR2, framework (FR) 3 and FR4 of the heavy chain. These differences aretoo numerous to be attributable to PCR or sequencing errors and haveprobably accrued during rounds of somatic mutation as the mouse wasrepeatedly boosted with antigen. The light chain sequences were alsovery similar, but with localized heterogeneity throughout the variabledomain, again probably resultant of somatic mutation.

Example 10

Selection of Anti-prion Antibodies Following Masking of Epitopes withExisting Antibodies

Panning of the IgG1 library against denatured PrP produced a series ofrelated antibodies, presumably somatic variants of a clone directed to asingle epitope (Example 9). To access antibodies to other epitopes, aprototype antibody from the above series was added to denatured PrP inELISA wells prior to panning in the normal way. The masking antibody wasused in all subsequent panning steps. Using this procedure, antibodieswere derived of different sequence which reacted with denatured PrP inELISA. These antibodies are likely directed to different epitopes onPrP. The masking procedure was carried out as described in Ditzel, et al(1995) J. Immunol. Masking could also be carried out with moleculesother than antibodies which interacted with PrP.

Example 11

Selection of Phage Particles Expressing Anti-PrP Antibodies Specific forPrP^(Sc)

A phage display antibody library similar to that described in theExamples above is subjected to panning experiments to identify phageclones that bind to PrP^(Sc), but not to PrP^(C). PrP^(Sc) antigen andPrP^(C) antigen are bound to separate wells of a microtiter dish asdescribed above for the ELISA assay. The phage display antibody libraryis first panned over the PrP^(C) ELISA wells. Unbound phage areretrieved from the wells and pooled. Phage that binds to the PrP^(C)antigen are removed from the wells and either discarded or pooled forlater analyses. The pooled unbound phage are then again added to PrP^(C)ELISA wells, with selection again being based upon lack of binding tothe PrP^(C). After several repeated selections on the PrP^(C) antigen,the phage are pooled and panned on the ELISA wells containing thePrP^(Sc) antigen. The panning is repeated for several rounds, with thephage that binds to the PrP^(Sc) antigen being the phage that isselected for further rounds of panning. After 5 to 10 rounds of panningon the PrP^(Sc) antigen, the phage are isolated one from another. Theability of the PrP^(Sc)-specific specific phage or isolated Fab to bindPrP^(C) antigen can be double-checked by ELISA with the PrP^(C) antigen.The resulting selected phage are those that bind PrP^(Sc), but do notbind PrP^(C).

Example 12

Selection of Phage Particles Expressing Anti-PrP Antibodies to IdentifyPrP^(Sc) Regardless of Isoform

A phage display antibody library is prepared as described above fromlymphocyte RNA from a mouse immunized with several PrP^(Sc) isoforms, orfrom a pool of lymphocyte RNA from several mice immunized with differentPrP^(Sc) isoforms. The phage are then panned with several differentwells containing antigens from different isoforms of PrP^(Sc). The phageare panned over each PrP^(Sc) isoform with the selection being for phagethat bind the isoform at each stage. The phage are panned for a total ofabout 5 to 10 rounds on each PrP^(Sc) isoform. The phage that remainafter all stages of panning against all the isoforms tested are thenisolated. The immunoreactivity of each selected phage or isolated Fab istested by ELISA or Western blot or histochemistry against each of thevarious PrP^(Sc) isoforms, as well as for cross-reactivity with PrP^(C).

Example 13

Selection of Phage Particles Expressing Anti-PrP Antibodies Specific forIsoforms of PrP^(Sc)

A phage display antibody library prepared from lymphocyte RNA of a mouseimmunized with a specific PrP^(Sc) isoform is prepared according to theExamples above. The resulting phage are then selected for their abilityto bind only one specific PrP^(Sc) isoform by panning. The panning usesseveral different wells containing antigens from different isoforms ofPrP^(Sc), including one set of wells containing antigens from thespecific PrP^(Sc) isoform against which specific antibodies are desired.The phage are first panned over the undesirable PrP^(Sc) isoforms, withthe selection being for phage that do not bind the antigen. Panningcontinues for a total of about 5 to 10 rounds on each of the PrP^(Sc)isoforms. The phage that did not bind the undesirable PrP^(Sc) isoformsare then panned for about 5 to 10 rounds against the desirable PrP^(Sc)isoform, with selection for antigen binding. The phage that remain afterall rounds of panning are isolated. These selected phage are those thatexpress antibodies with binding specificity for only the specificPrP^(Sc) isoform desired. The immunoreactivity of each selected phage orisolated Fab is tested by ELISA or Western blot against each of thevarious PrP^(Sc) isoforms, as well as for cross-reactivity with PrP^(C).

Example 14

Generation and Characterization Of Serum Reactivity Against PrP^(Sc) InPrP^(0/0) Mice

Experimentation per the above Examples established that the primaryprognostic indicator for success in isolating a specific antibody fromcombinatorial libraries with the size range of 10⁷ pfu/ml is the serumreactivity with the antigen to be studied, and it is this factor whichwill ultimately dictate the composition of the library. AlthoughPrnp^(0/0) mice elucidated a strong immune response upon immunizationwith either mouse (Mo) or Syrian hamster (SHa) prion rods composed ofPrP 27-30 proteins, the highest serum titers were seen in the IgG1 andIgG2b subclasses. The IgG2a and IgG3 anti-PrP titers were close to thebackground levels of reactivity seen for all IgG subclasses in the serumof non-immunized mice. In an attempt to increase the immune response andaugment the immune repertoire against PrP^(Sc), Prnp^(0/0) (94% FVB)female mice were immunized with liposomes containing SHaPrP 27-30. Tofurther increase the immune response diversity, mice were immunizedusing both short and long term protocols. In contrast to immunizationwith SHa prion rods immunization with liposomes containing SHaPrP 27-30resulted in antiserum titer which includes all four IgG subclasses.

Example 15

PrP-immunized Sera Reactivity Against Histoblots

To further investigate the properties of the IgG anti-SHaPrP 27-30 foundin the sera from mice immunized with liposomes containing SHaPrP 27-30,we tested the sera in situ with histoblotting techniques, in whichcryostat sections of normal and scrapie infected SHa brain weretransferred onto nitrocellulose membranes. Although both sera showedsome nonspecific reactivity against proteinase K (PK)-treated normal SHabrain sections, only the sera from the long term immunized mice showedincreased reactivity against PK-treated SHa scrapie infected brainsections. This reactivity was also evident in sera dilution to 1/1000(results not shown). Both sera showed typical reactivity against SHascrapie infected brain sections which were first PK-treated and thenexposed to 3M GdnSCN for 10 minutes. Sera from non-immunized Prnp^(0/0)(946 FVB) female mice did not show any immune reactivity against normalscrapie infected SHa brain sections. Staining of SHaPrP 27-30 andDenatured SHaPrP 27-30 in Histoblots of Scrapie Infected SHa BrainHistoblots were treated with proteinase K to remove PrP^(C) from thebrain of normal, uninoculated control SHa and SHa showing clinical signsof scrapie following inoculation with Sc237 prions. To denature SHaPrP27-30, histoblots were treated with 3M GdnSCN for 10 minutes. Blots wereincubated overnight at 4° C. with sera diluted {fraction (1/200)} fromthe short and the long term immunized mice. The results described hereshow clear positive reactivity of an antiserum with non-denaturedinfectious prions i.e., native PrP^(Sc).

FIG. 8 shows eight different stained histoblots of scrapie infected SHabrain. The histoblots were treated with proteinase K to remove PrP^(C)from the brain of normal, non-inoculated control SHa(A, C, E and G) andSHa showing clinical signs of scrapie following inoculation with Sc 237prions (B, D, F and H). To denature the SHaPrP 27-30, the histoblotswere treated with 3M GdnSCN for 10 minutes (C, D, G and H). The blotswere incubated overnight at 4° C. with sera diluted {fraction (1/200)}from the short (A-D) and the long (E-H) term immunized mice. The resultsclearly show the ability of the antibodies of the invention to bind tonative, non-denatured infectious prions i.e., bind to native PrP^(Sc).

Example 16

Generation Of Monoclonal Antibodies From Immunized Mice Of Example 14

Overall, eight phage Fab display libraries were constructed: IgG1k,IgG2ak, IgG2bk and IgG3k from mRNA extracted from the short and longterm immunized mice. To overcome difficulties with the isolation ofphage expressing anti-PrP Fab by panning against prion rods containingPrP 27-30, a panning system was used where libraries are panned againstbiotinylated SHa 27-30, dispersed into liposomes, and bound tostreptavidin-coated microtiter plates. After five rounds of panning, E.Coli extracts from more than 50 clones reacted with biotinylated SHa27-30, SHa 27-30 rods and 90-231 recombinant SHa in ELISA. Since theseclones also react with recombinant rPrP corresponding to SHaPrP residues90-231, Melhorn,I., et al, High-level Expression and Characterization ofa Purified 142-residue Polypeptide of the Prion Protein. Biochemistry35, 5528-2237 (1996), all eight libraries were panned against thisantigen to successfully isolate more distinct clones from virtually allthe libraries. Upon DNA sequencing of the plasmid region coding for theIgG heavy chain, 30 Fabs were identified as distinct clones.

Example 17

Characterization Of Monoclonal Antibodies

Initial ELISA with E. Coli extracts from positive clones suggested thatthe Fabs, in contrast to the monoclonal 3F4 antibody, Kascsak, R. J., etal, Mouse Polyclonal and Monoclonal Antibody to Scrapie AssociatedFibril Proteins, J. Virol. 61, 3688-3693 (1987), bind to PrP 27-30 in anative state, i.e., without a denaturation step. To characterizequantitatively the novelty of these Fabs, we purified them and produced3F4 Fab from the monoclonal 3F4 by enzymatic cleavage. Standard ELISAfor the detection of SHaPrP was performed using different concentrationsof the purified Fabs. In contrast to 3F4 which showed characteristic SHaPrP binding properties (basal binding to prion rods and strongreactivity against SHaPrP 27-30 after treatment with 3M non-denaturantGdnSCN), the newly isolated Fabs reacted against prion rods without anydenaturation step. The half-maximal binding to non-denatured prion rodsoccurs at a Fab concentration of approximately 0.5 pg/ml, indicatingthat the antibody has an apparent binding affinity of approximately 10⁸moles/liter.

FIG. 9 is a graph showing the ELISA reactivity of purified Fabs againstprion protein SHa 27-30. The antibody 3F4 and recombinant antibodieswere examined at different concentrations for binding to ELISA wellswhich were coated with 0.2 μ/g of sucrose purified infectious SHa prionrods. The results clearly show that all of the recombinant antibodies ofthe invention have substantially higher degrees of binding to prions ascompared to the antibody 3F4.

Protocol For ELISA Reactivity Of Purified Fabs Against Denatured PrionProtein SHa 27-30

Purified 3F4 Fab and recombinant Fabs were examined at differentconcentrations for binding to ELISA wells coated with 0.2 μg of sucrosepurified SHa prion rods either native or denatured in the ELISA wellwith 3M GdnSCN for 10 min.

FIG. 10 is a graph showing the results of ELISA reactivity purified Fabsagainst denatured prion protein SHa 27-30. FIG. 10 is interesting ascompared to FIG. 9 in that the recombinant antibodies of the inventionas per FIG. 9 show a higher degree of affinity for the prion rods ascompared to 3F4 whereas all of the recombinant antibodies but for R1show a lower degree of affinity against denatured antigen.

Example 18 Characterization Of Monoclonal Antibody ByImmunoprecipitation

Immunoprecipitation of SHaPrP 27-30

To confirm the anti-PrP 27-30 activity of the Fabs as well as to confirmthe in-ability of 3F4 to bind nondenatured SHaPrP 27-30, animmunoprecipitation method was developed using liposomes containing SHa27-30. E. Coli extracts from Fab producing clones immunoprecipitated40-50% of the SHaPrP 27-30 present in the solution, while 3F4 indilution of 1/500 immunoprecipitated only trace amounts of SHaPrP. Fabconcentrations in bacterial supernates are typically on the order of1-10 pg/ml. This implies that the affinity for antigen are high (on theorder of 10⁷-10⁸ moles/liter or more). The antibody 3F4 was obtained asan ascetic fluid and is expected to have a concentration ofapproximately 1 pg/ml at the dilution used in the immunoprecipitationexperiment. The ability of the new Fabs to immunoprecipitate SHaPrP27-30 in comparison to 3F4 was determined quantitatively with purifiedFab mAbs D4 and R2. Fab 2R immunoprecipitated SHaPrP 27-30 strongly atconcentrations as low as 0.1 pg/ml (50 ng in 500 pl) indicating anaffinity on the order of greater than 10⁸M⁻¹ (i.e., 10⁸ mole/liter). Fab2R was less potent but clearly immune precipitated antigen moreefficiently than 3F4. Note that D4, R2, 6D2, D14, R1, and R10 all referto antibodies of the invention.

Immunoprecipitation of SHaPrP 27-30 with Recombinant Fabs The ability of3F4 diluted 1/500 and 100 μl of E. Coli extracts containing Fab toimmunoprecipitate SHaPrP 27-30 was monitored by western blotting. Alllanes except lane 14 are from immunoprecipitations containing goatanti-mouse IgG Fab and protein A agarose. 10 μl of liposomes containingSHa PrP 27-30 were added to lanes 1, 3, 5, 7, 9, 11, 13. 100 μl of E.Coli extracts from different clones diluted 1/500 were added as follows:lanes 2-3, 6D2; lanes 4-5, D14; lanes 6-7, R1; lanes 8-9, R10; lanes10-11, D4; lanes 12-13, 3F4. Lane 14 was loaded with ½ volume ofliposomes used for immunoprecipitations.

The results described above are shown within the photograph of FIG. 11.The photo clearly shows higher degrees of immunoprecipitation when usingthe recombinant antibodies of the invention.

FIG. 12 is a photo showing the immunoprecipitation of SHaPrP 27-30 withpurified Fabs of the invention (2R and 4D) as well as 3H4. The abilityto immunoprecipitate the antigen is monitored by western blotting. Allof the lanes shown in FIG. 12 but for lane 14 are immunoprecipitationscontaining goat anti-mouse IgG Fab and protein Agarose. To obtain theresults 10 μl of liposomes containing SHaPrP 27-30 were added to alllanes except for lanes 5, 9 and 13. Each of the lanes are marked withthe indicated amounts of purified Fabs (nanograms) which were added tolanes 2-13. Lane 14 was loaded with one-half volume of liposomes usedfor the immunoprecipitation. The results clearly show a dramaticallyhigher degree of precipitation when using the antibodies 2R and 4D ofthe invention as compared to 3F4.

The ELISA data (FIG. 9) clearly show a number of Fabs with a saturablebinding to non-denatured PrP 27-30 and a half-maximal binding at around0.5 μg/ml. This corresponds to an apparent affinity constant at 10⁸ M⁻¹(MW of Fab=50,000). At the same time, 3F4 shows insignificant bindingout to 2 μg/ml. Moving to denatured PrP 27-30, FIG. 10, the recombinantFabs now bind to a higher level but with a similar apparent affinity.This suggests denaturation has revealed more antigenic sites but theiraffinities are the same. Significantly, 3F4 is now binding comparably tothe recombinant Fabs with an apparent affinity of the order of 10⁸ M⁻¹.Comparison of the 3F4 data in FIGS. 9 and 10 strongly suggests theintegrity of PrP 27-30 in the non-denatured form. Thus it could havebeen argued that the recombinant Fabs were reacting with a fraction ofdenatured PrP present in the PrP 27-30 preparation. The lack ofreactivity of 3F4 with non-denatured PrP 27-30 coupled with its strongreactivity with denatured PrP 27-30 refutes this interpretation andstrongly suggests the recombinant Fabs recognize non-denatured rods withhigh affinity.

The immunoprecipitation data are confirmatory of the ELISA data. Lowconcentrations of recombinant Fabs as found in crude bacterialsupernates (typically 1-10 μl/ml) are highly effective atimmunoprecipitating PrP 27-30 (FIG. 11). This implies an affinity on theorder of 10⁷-10⁸ M⁻¹. Under comparable concentration conditions, 3F4does not produce significant precipitation. A more quantitative analysis(FIG. 12) shows that Fab R2 immunoprecipitates PrP 27-30 highlyeffectively with some titration in the range 0.1-0.2 μg/ml implying abinding affinity on the order of 10⁸ M⁻¹. Fab 4D has a lower affinityand 3F4 immunoprecipitates very weakly indeed. From this particularexperiment one could argue that the affinity of 3F4 is considerably lessthan 5×10⁷ M⁻¹ and probably less than 10⁷ M⁻¹.

Overall, the data indicates that the recombinant Fabs have affinities inthe range of 10⁷-10⁸ M⁻¹.

The instant invention is shown and described herein in what isconsidered to be a most practical and preferred embodiments. It isrecognized, however, that departures may be made from which are withinthe scope of S the invention and that modifications will occur to onewho is skilled in the art upon reading this disclosure.

86 254 amino acids amino acid single linear peptide not provided 1 MetAla Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25 30Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 40 45Tyr Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly Gly Gly Trp 50 55 60Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Ser Trp 65 70 7580 Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 85 9095 Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Leu Lys His Val Ala 100105 110 Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met115 120 125 Leu Gly Ser Ala Met Ser Arg Pro Met Ile His Phe Gly Asn AspTrp 130 135 140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro AsnGln Val 145 150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln AsnAsn Phe Val His 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His ThrVal Thr Thr Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp ValLys Met Met Glu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln TyrGln Lys Glu Ser Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser SerThr Val Leu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile SerPhe Leu Ile Phe Leu Ile Val Gly 245 250 253 amino acids amino acidsingle linear peptide not provided 2 Met Ala Asn Leu Gly Cys Trp Met LeuVal Leu Phe Val Ala Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys LysArg Pro Lys Pro Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro GlyGln Gly Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly GlyTrp Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly GlyGly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro SerLys Pro Lys Thr Asn Met Lys His Met 100 105 110 Ala Gly Ala Ala Ala AlaGly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 Met Leu Gly Ser AlaMet Ser Arg Pro Ile Ile His Phe Gly Ser Asp 130 135 140 Tyr Glu Asp ArgTyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 Val TyrTyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175 HisAsp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180 185 190Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg 195 200205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245250 263 amino acids amino acid single linear peptide not provided 3 MetVal Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 1 5 10 15Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly 35 40 45Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly 50 55 60Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 65 70 7580 Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 85 9095 Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys Pro 100105 110 Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala Ala115 120 125 Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser AlaMet 130 135 140 Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp ArgTyr Tyr 145 150 155 160 Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val TyrTyr Arg Pro Val 165 170 175 Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val HisAsp Cys Val Asn Ile 180 185 190 Thr Val Lys Glu His Thr Val Thr Thr ThrThr Lys Gly Glu Asn Phe 195 200 205 Thr Glu Thr Asp Ile Lys Met Met GluArg Val Val Glu Gln Met Cys 210 215 220 Val Thr Gln Tyr Gln Lys Glu SerGln Ala Tyr Tyr Asp Gln Gly Ala 225 230 235 240 Ser Val Ile Leu Phe SerSer Pro Pro Val Ile Leu Leu Ile Ser Phe 245 250 255 Leu Ile Phe Leu IleVal Gly 260 255 amino acids amino acid single linear peptide notprovided 4 Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe ValAla 1 5 10 15 Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys ProGly Gly 20 25 30 Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser ProGly Gly 35 40 45 Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln ProHis Gly 50 55 60 Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln ProHis Gly 65 70 75 80 Gly Ser Trp Gly Gln Pro His Gly Gly Gly Gly Trp GlyGln Gly Gly 85 90 95 Ser His Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys ThrAsn Met Lys 100 105 110 His Val Ala Gly Ala Ala Ala Ala Gly Ala Val ValGly Gly Leu Gly 115 120 125 Gly Tyr Met Leu Gly Ser Ala Met Ser Arg ProLeu Ile His Phe Gly 130 135 140 Asn Asp Tyr Glu Asp Arg Tyr Tyr Arg GluAsn Met Tyr Arg Tyr Pro 145 150 155 160 Asn Gln Val Tyr Tyr Arg Pro ValAsp Gln Tyr Ser Asn Gln Asn Asn 165 170 175 Phe Val His Asp Cys Val AsnIle Thr Val Lys Gln His Thr Val Thr 180 185 190 Thr Thr Thr Lys Gly GluAsn Phe Thr Glu Thr Asp Ile Lys Ile Met 195 200 205 Glu Arg Val Val GluGln Met Cys Ile Thr Gln Tyr Gln Arg Glu Ser 210 215 220 Gln Ala Tyr TyrGln Arg Gly Ala Ser Val Ile Leu Phe Ser Ser Pro 225 230 235 240 Pro ValIle Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 255 27 basepairs nucleic acid single linear cDNA not provided 5 CAGGTGCAGCTCGAGGAGTC AGGACCT 27 27 base pairs nucleic acid single linear cDNA notprovided 6 GAGGTGCAGC TCGAGGAGTC AGGACCT 27 27 base pairs nucleic acidsingle linear cDNA not provided 7 GAGGTCCAGC TCGAGCAGTC TGGACCT 27 27base pairs nucleic acid single linear cDNA not provided 8 CAGGTCCAACTCGAGCAGCC TGGGGTC 27 28 base pairs nucleic acid single linear cDNA notprovided 9 GAGGTTCAGC TCGAGCAGTC TGGGGCAA 28 27 base pairs nucleic acidsingle linear cDNA not provided 10 GAAGTGAAGC TCGAGGAGTC TGGAGGA 27 27base pairs nucleic acid single linear cDNA not provided 11 GAGGTGAAGCTCGAGGAGTC TGGAGGA 27 27 base pairs nucleic acid single linear cDNA notprovided 12 GAGGTGAAGC TTCTCGAGTC TGGAGGT 27 27 base pairs nucleic acidsingle linear cDNA not provided 13 GAAGTGAAGC TCGAGGAGTC TGGGGGA 27 30base pairs nucleic acid single linear cDNA not provided 14 GAGGTTCAGCTCGAGGAGCA GTCTGGAGCT 30 22 base pairs nucleic acid single linear cDNAnot provided 15 AGGTCCAGCT GCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 16 AGGTGCAGCT GCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 17 AGGTCAAGCTGCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 18 AGGTGAAGCT GCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 19 AGGTCCAACT GCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 20 AGGTGCAACTGCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 21 AGGTCAAACT GCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 22 AGGTGAAACT GCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 23 AGGTCCAGCTTCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 24 AGGTGCAGCT TCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 25 AGGTCAAGCT TCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 26 AGGTGAAGCTTCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 27 AGGTCCAACT TCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 28 AGGTGCAACT TCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 29 AGGTCAAACTTCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 30 AGGTGAAACT TCTCGAGTCT GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 31 AGGTCCAGCT GCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 32 AGGTGCAGCTGCTCGAGTCA GG 22 22 base pairs nucleic acid single linear cDNA notprovided 33 AGGTCAAGCT GCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 34 AGGTGAAGCT GCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 35 AGGTCCAACTGCTCGAGTCA GG 22 22 base pairs nucleic acid single linear cDNA notprovided 36 AGGTGCAACT GCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 37 AGGTCAAACT GCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 38 AGGTGAAACTGCTCGAGTCA GG 22 22 base pairs nucleic acid single linear cDNA notprovided 39 AGGTCCAGCT TCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 40 AGGTGCAGCT TCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 41 AGGTCAAGCTTCTCGAGTCA GG 22 22 base pairs nucleic acid single linear cDNA notprovided 42 AGGTGAAGCT TCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 43 AGGTCCAACT TCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 44 AGGTGCAACTTCTCGAGTCA GG 22 22 base pairs nucleic acid single linear cDNA notprovided 45 AGGTCAAACT TCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 46 AGGTGAAACT TCTCGAGTCA GG 22 22 basepairs nucleic acid single linear cDNA not provided 47 AGGTCCAGCTGCTCGAGTCT GG 22 22 base pairs nucleic acid single linear cDNA notprovided 48 AGGTCCAGCT GCTCGAGTCA GG 22 22 base pairs nucleic acidsingle linear cDNA not provided 49 AGGTCCAGCT TCTCGAGTCT GG 22 22 basepairs nucleic acid single linear cDNA not provided 50 AGGTCCAGCTTCTCGAGTCA GG 22 32 base pairs nucleic acid single linear cDNA notprovided 51 CCAGTTCCGA GCTCGTTGTG ACTCAGGAAT CT 32 32 base pairs nucleicacid single linear cDNA not provided 52 CCAGTTCCGA GCTCGTGGTG ACGCAGCCGCCC 32 32 base pairs nucleic acid single linear cDNA not provided 53CCAGTTCCGA GCTCGTGCTC ACCCAGTCTC CA 32 32 base pairs nucleic acid singlelinear cDNA not provided 54 CCAGTTCCGA GCTCCAGATG ACCCAGTCTC CA 32 29base pairs nucleic acid single linear cDNA not provided 55 CCAGATGTGAGCTCGTGACC CAGACTCCA 29 32 base pairs nucleic acid single linear cDNAnot provided 56 CCAGATGTGA GCTCGTCATG ACCCAGTCTC CA 32 32 base pairsnucleic acid single linear cDNA not provided 57 CCAGTTCCGA GCTCGTGATGACACAGTCTC CA 32 34 base pairs nucleic acid single linear cDNA notprovided 58 GCGCCGTCTA GAATTAACAC TCATTCCTGT TGAA 34 22 base pairsnucleic acid single linear cDNA not provided 59 AGGTCCAACT GCTCGAGTCT GG22 22 base pairs nucleic acid single linear cDNA not provided 60AGGTCCAACT GCTCGAGTTC AG 22 22 base pairs nucleic acid single linearcDNA not provided 61 AGGTCCAACT TCTCGAGTCT GG 22 30 base pairs nucleicacid single linear cDNA not provided 62 AGGCTTACTA GTACAATCCC TGGGCACAAT30 27 base pairs nucleic acid single linear cDNA not provided 63CTCCTTACTA GTAGGACAGG GGATTGT 27 18 base pairs nucleic acid singlelinear cDNA not provided 64 CACGACTGAG GCACCTCC 18 22 base pairs nucleicacid single linear cDNA not provided 65 AAGACAGCTA TCGCGATTGC AG 22 30base pairs nucleic acid single linear cDNA not provided 66 ATAGCCCTTGACCAGGCATC CCAGGGTCAC 30 21 base pairs nucleic acid single linear cDNAnot provided 67 ACCTATTGCC TACGGCAGCC G 21 114 amino acids amino acidsingle linear peptide not provided 68 Leu Glu Gln Ser Gly Val Glu LeuAla Arg Pro Gly Ala Ser Val Met 1 5 10 15 Leu Ser Cys Lys Ala Ser GlyTyr Thr Phe Thr Thr Tyr Gly Ile Ser 20 25 30 Trp Val Lys Gln Arg Thr GlyGln Gly Leu Glu Trp Ile Gly Glu Ile 35 40 45 Trp Pro Arg Ser Gly Asn ThrTyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 55 60 Ala Thr Leu Thr Ala Asp LysSer Ser Ser Thr Ala Tyr Leu Asp Leu 65 70 75 80 Arg Ser Leu Thr Ser GluAsp Ser Ala Val Tyr Phe Cys Ala Arg His 85 90 95 Asp Gly Tyr Pro Phe AlaTyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ala 114 aminoacids amino acid single linear peptide not provided 69 Leu Glu Gln SerGly Val Glu Leu Ala Arg Pro Gly Ala Ser Val Met 1 5 10 15 Leu Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser 20 25 30 Trp Val LysGln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 35 40 45 Cys Pro ArgSer Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 55 60 Ala Thr LeuThr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu 65 70 75 80 Arg SerLeu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His 85 90 95 Asp GlyTyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 SerAla 91 amino acids amino acid single linear peptide not provided 70 TyrThr Phe Thr Thr Tyr Gly Ile Thr Trp Val Lys Gln Arg Thr Gly 1 5 10 15Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr 20 25 30Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys 35 40 45Ser Ser Ser Thr Ala Tyr Met Glu Val Arg Ser Leu Thr Ser Asp Asp 50 55 60Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr 65 70 7580 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 85 90 91 amino acidsamino acid single linear peptide not provided 71 Xaa Thr Phe Thr Val TyrGly Ile Ser Trp Val Lys Gln Arg Thr Gly 1 5 10 15 Gln Gly Leu Glu TrpIle Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr 20 25 30 Tyr Tyr Asn Glu LysPhe Lys Val Lys Ala Thr Leu Ser Ala Asp Lys 35 40 45 Ser Ser Ser Thr AlaSer Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 50 55 60 Ser Ala Val Tyr PheCys Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr 65 70 75 80 Trp Gly Gln GlyThr Leu Val Thr Val Ser Ala 85 90 95 amino acids amino acid singlelinear peptide not provided 72 Trp Glu Xaa Arg Val Ser Leu Thr Cys ArgAla Ser Gln Asp Phe Gly 1 5 10 15 Ser Ser Leu Asn Trp Phe Arg Gln LysPro Asp Gly Thr Ile Arg Arg 20 25 30 Leu Ile Tyr Ala Thr Ser Arg Leu HisSer Gly Val Pro Lys Arg Phe 35 40 45 Ser Gly Ser Arg Ser Gly Ser Asp TyrSer Leu Thr Ile Ser Ser Leu 50 55 60 Glu Ala Glu Asp Phe Gly Asp Tyr TyrCys Leu Gln Tyr Ala Ala Ser 65 70 75 80 Pro Phe Thr Phe Gly Ser Gly ThrLys Leu Glu Ile Lys Arg Ala 85 90 95 109 amino acids amino acid singlelinear peptide not provided 73 Glu Leu Val Met Thr Gln Thr Pro Ser SerLeu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg AlaSer Gln Asp Phe Gly Ser Ser 20 25 30 Leu Asn Trp Phe Arg Gln Ala Pro AspGly Thr Ile Arg Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Lys Leu His Ser GlyVal Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp His Ser LeuThr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Leu Gly Asn Tyr Tyr CysLeu Gln Tyr Ala Ala Ser Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys LeuGlu Ile Lys Arg Ala 100 105 109 amino acids amino acid single linearpeptide not provided 74 Glu Leu Gln Met Thr Gln Thr Pro Ser Ser Leu SerVal Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser GlnAsp Ile Gly Ser Ser 20 25 30 Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly ThrIle Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val ProLys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr IleSer Ser Leu Glu Ser 65 70 75 80 Glu Asp Leu Val Asp Tyr Tyr Cys Leu GlnTyr Ala Ser Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu IleLys Arg Ala 100 105 105 amino acids amino acid single linear peptide notprovided 75 Xaa Leu Gly Arg Gln Val Met Leu Ser Ser Lys Ala Ser Xaa TyrThr 1 5 10 15 Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr GlyGln Gly 20 25 30 Leu Glu Trp Ile Gly Glu Ile Cys Pro Arg Ser Gly Asn ThrTyr Tyr 35 40 45 Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp LysSer Ser 50 55 60 Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu Thr Ser Glu AspSer Ala 65 70 75 80 Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe AlaTyr Trp Gly 85 90 95 Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 114amino acids amino acid single linear peptide not provided 76 Leu Glu GlnSer Gly Val Glu Leu Ala Arg Pro Gly Xaa Ser Val Lys 1 5 10 15 Leu SerCys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Thr 20 25 30 Trp ValLys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 35 40 45 Trp ProArg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 55 60 Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Val 65 70 75 80 ArgSer Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys Ala Arg His 85 90 95 AspGly Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110Ser Ala 114 amino acids amino acid single linear peptide not provided 77Leu Glu Gln Ser Gly Val Glu Leu Ala Gly Pro Gly Ala Ser Val Lys 1 5 1015 Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser 20 2530 Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 35 4045 Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 5560 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu 65 7075 80 Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His 8590 95 Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val100 105 110 Ser Ala 91 amino acids amino acid single linear peptide notprovided 78 Xaa Thr Phe Thr Thr Tyr Gly Ile Thr Trp Val Lys Gln Arg ThrGly 1 5 10 15 Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser GlyAsn Thr 20 25 30 Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr AlaAsp Lys 35 40 45 Ser Ser Ser Thr Ala Tyr Met Glu Val Arg Ser Leu Thr SerAsp Asp 50 55 60 Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro PheAla Tyr 65 70 75 80 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 85 90 92amino acids amino acid single linear peptide not provided 79 Xaa Tyr ThrPhe Thr Thr Tyr Gly Ile Thr Trp Val Lys Gln Arg Thr 1 5 10 15 Gly GlnAsp Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn 20 25 30 Thr TyrTyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Ala Ala Asp 35 40 45 Lys SerSer Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Asp 50 55 60 Asp SerAla Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala 65 70 75 80 TyrTrp Asp Gln Gly Thr Leu Val Thr Val Ser Thr 85 90 99 amino acids aminoacid single linear peptide not provided 80 Xaa Leu Ser Cys Lys Ala SerGly Tyr Thr Phe Thr Val Tyr Gly Ile 1 5 10 15 Ser Trp Val Lys Gln ArgThr Gly Gln Gly Leu Glu Trp Ile Gly Glu 20 25 30 Ile Trp Pro Arg Ser GlyAsn Thr Tyr Tyr Asn Glu Lys Phe Lys Val 35 40 45 Lys Ala Thr Leu Thr AlaAsp Lys Ser Ser Ser Thr Ala Ser Met Glu 50 55 60 Leu Arg Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Phe Cys Ala Arg 65 70 75 80 His Asp Gly Tyr ProPhe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 85 90 95 Val Ser Ala 91amino acids amino acid single linear peptide not provided 81 Xaa Thr PheThr Val Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr Gly 1 5 10 15 Gln GlyLeu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr 20 25 30 Tyr TyrAsn Glu Lys Phe Lys Val Lys Ala Thr Leu Thr Xaa Asp Lys 35 40 45 Ser SerSer Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 50 55 60 Ser AlaVal Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr 65 70 75 80 TrpGly Gln Gly Thr Leu Val Thr Val Ser Thr 85 90 101 amino acids amino acidsingle linear peptide not provided 82 Ser Val Lys Leu Ser Cys Lys AlaSer Gly Tyr Thr Phe Thr Thr Tyr 1 5 10 15 Gly Ile Ser Trp Val Lys GlnArg Thr Gly Gln Gly Leu Glu Trp Ile 20 25 30 Gly Glu Ile Trp Pro Arg SerGly Asn Thr Tyr Tyr Asn Glu Lys Phe 35 40 45 Lys Gly Lys Ala Thr Leu SerAla Asp Lys Ser Ser Ser Thr Ala Tyr 50 55 60 Leu Asp Leu Arg Ser Leu ThrSer Glu Asp Ser Ala Val Tyr Phe Cys 65 70 75 80 Ala Arg His Asp Gly TyrPro Phe Ala Tyr Trp Gly Gln Gly Thr Leu 85 90 95 Val Thr Val Ser Ala 100108 amino acids amino acid single linear peptide not provided 83 Glu LeuXaa Xaa Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser 1 5 10 15 GlyXaa Thr Phe Thr Thr Tyr Gly Ile Thr Trp Val Lys Gln Arg Thr 20 25 30 GlyGln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn 35 40 45 ThrTyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp 50 55 60 LysSer Ser Ser Thr Ala Tyr Met Glu Val Arg Ser Leu Thr Ser Asp 65 70 75 80Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala 85 90 95Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 103 amino acidsamino acid single linear peptide not provided 84 Pro Gly Pro Ser Val LysLeu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 1 5 10 15 Thr Thr Tyr Gly IleSer Trp Val Lys Gln Arg Thr Gly Gln Gly Leu 20 25 30 Glu Trp Ile Gly GluIle Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn 35 40 45 Glu Lys Phe Lys GlyLys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 50 55 60 Thr Ala Tyr Leu AspLeu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 65 70 75 80 Tyr Phe Cys AlaArg His Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln 85 90 95 Gly Thr Leu ValThr Val Ser 100 92 amino acids amino acid single linear peptide notprovided 85 Xaa Asn Thr Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys Gln ArgThr 1 5 10 15 Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg SerGly Asn 20 25 30 Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu ThrAla Asp 35 40 45 Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu ThrSer Glu 50 55 60 Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr ProPhe Ala 65 70 75 80 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 8590 95 amino acids amino acid single linear peptide not provided 86 XaaAla Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys 1 5 10 15Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg 20 25 30Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu 35 40 45Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu 50 55 60Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr 65 70 7580 Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 85 90 95

What is claimed is:
 1. A method of detecting PrP^(Sc) in a source comprising: contacting a source suspected of containing native PrP^(Sc) with a diagnostically effective amount of an antibody characterized by its ability to bind to native PrP^(Sc) in situ; and determining whether the antibody binds specifically to any material in the source.
 2. The method of claim 1, wherein the antibody binds to native PrP^(Sc) from a mammal selected from the group consisting of a human, a cow, a sheep, a horse, a pig, a dog, a chicken and a cat.
 3. The method of claim 1, wherein the antibody binds to said native PrP^(Sc) with a binding, affinity K_(a) of 10⁷ l/mol or more.
 4. The method of claim 3, wherein the K_(a) is 10⁸ l/mol or more.
 5. The method of claim 1, wherein the antibody is attached to a detectable label and the detecting is in vivo.
 6. The method of claim 1, wherein the antibody is attached to a detectable label, and, wherein the label is selected from a group consisting of a radioisotope label and a paramagnetic label; and wherein the antibody is attached to a substrate and the detecting is in vitro.
 7. An assay, comprising: a support surface; and an antibody bound to the surface of the support, the antibody characterized by an ability to bind native PrP^(Sc) in situ with a binding affinity k_(a) of 10⁷ l/mol or more.
 8. The assay of claim 7, wherein the antibody is further characterized by an ability to bind 50% or more of native PrP^(Sc) in a liquid flowable sample.
 9. The assay of claim 7, wherein a plurality of different antibodies are bound to the support surface and each antibody has a binding affinity K_(a) of 10⁷ l/mol or more to native PrP^(Sc). 